Acellular pro-tolerogenic compositions for the treatment/prevention of auto-immune diseases

ABSTRACT

This invention relates to pro-tolerogenic preparations capable of increasing the level of regulatory T cells (Treg) and/or decreasing the level of pro-inflammatory T cells (Th17) to induce anergy or immune tolerance. The pro-tolerogenic preparation is enriched in at least one species of miRNAs and is obtained by contacting two allogeneic leukocyte populations wherein at least one of the two populations is modified with a low-immunogenic biocompatible polymer. Therapeutic uses intended for the treatment/prevention of auto-immune diseases of such compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/CA2013/050544 filed Jul. 12, 2013,which claims priority from CA patent application 2782942, U.S.provisional patent application 61/670,636 and U.S. provisional patentapplication 61/670,694, all filed on Jul. 12, 2012. The entire contentsof each of the above-referenced disclosures is specifically incorporatedby reference herein without disclaimer.

TECHNOLOGICAL FIELD

The invention provides pro-tolerogenic preparations capable of inducingimmune tolerance or anergy. The pro-tolerogenic preparations areRNase-sensitive and can be obtained by enriching the miRNA speciesexpressed when two allogeneic leukocyte populations are contacted and atleast one of the population has been modified to bear on its surface alow-immunogenic biocompatible polymer capable of limitingpro-inflammatory allo-recognition.

BACKGROUND

Acute and chronic rejection of donor tissues and organs remains asignificant clinical problem in transplantation medicine. Moreover,autoimmune diseases in which one's own immune system recognizes “self”tissues as foreign can also be rejected by similar mechanisms. Tominimize or prevent rejection, the administration of immunosuppressiveagents is typically required. Acute and chronic rejection are primarilyT lymphocyte-mediated events that require allogeneic recognition of theforeign tissue and the subsequent proliferation of allo-responsive Tcells. Indeed, because of the central role of the T cell in rejection,it is the primary target of current immunosuppressive drugs (e.g.,cyclosporine A, FK506). In general, these pharmacologic agents targeteither the T cell activation (e.g., cyclosporine A that inhibits IL-2responsiveness) or the proliferation (e.g., methotrexate) of theallo-responsive T cells. However all of today's clinically approvedanti-rejection drugs are beset by chronic toxicity. Consequently,significant research is underway to identify alternative means ofpreventing acute and chronic rejection.

A biomaterials approach to modulating immune responsiveness is thedirect modification of the surface of donor cells (e.g., erythrocytes,lymphocytes, and pancreatic islets) to prevent allo-recognition (Scottet al., 1997; Murad et al., 1999A; Murad et al., 1999B; Bradley et al.,2001; Chen et al., 2001; Chen et al., 2003; McCoy et al., 2005; Chen etal., 2006; Bradley et al., 2007; Sutton et al., 2010; Le et al., 2010).The polymer modification of the surface of cells is induced by thedirect grafting of low immunogenicity polymers to the cell membrane.Previous studies have demonstrated that the polymer modification of thesurface of erythrocytes and lymphocytes resulted in the loss ofallo-recognition both in vitro and in vivo. Moreover, in contrast topharmacologic agents, the grafted polymer exhibited both extremely lowtoxicity and immunogenicity.

It would be highly desirable to be provided with an acellularpreparation capable of inducing a state of anergy or immunotolerance byincreasing the ratio of the level of regulatory T cells (such as Treg)to pro-inflammatory T cells (such as Th1 and Th17). The acellularpreparation could induce anergy or tolerance either by increasing Treglevels, decreasing pro-inflammatory T cell levels or both. Thispreparation could be useful for treating, preventing and/or alleviatingthe symptoms associated to an abnormal/excessive immune condition, suchas an auto-immune disease, an exacerbated response to a vaccine or atissue/cell transplantation.

BRIEF SUMMARY

The present invention concern acellular preparations, enriched in atleast one miRNA species obtained by contacting two allogeneic leukocytepopulations, wherein one of the leukocyte population has been modifiedto bear on its surface a low-immunogenic biocompatible polymer. The twoleukocyte populations are contacted in conditions preventing or limitingpro-inflammatory allo-recognition while allowing pro-tolerogenicallo-recognition.

According to a first aspect, the present invention provides a processfor making an acellular pro-tolerogenic composition. Broadly, theprocess comprises (i) associating a low-immunogenic biocompatiblepolymer to a cytoplasmic membrane of a first leukocyte to obtain a firstmodified leukocyte; (ii) contacting the first modified leukocyte with asecond leukocyte under conditions to allow a pro-tolerogenicallorecognition to provide a conditioned preparation, wherein the firstleukocyte is allogeneic to the second leukocyte; (iii) removing thefirst modified leukocyte and the second leukocyte from the conditionedpreparation under conditions to inhibit RNA degradation so as to obtaina composition enriched in acellular pro-tolerogenic components; and (iv)formulating the composition of step (iii), under conditions to inhibitRNA degradation, in the acellular pro-tolerogenic preparation foradministration to a subject. In an embodiment, the process furthercomprises covalently binding the low-immunogenic biocompatible polymerto a membrane-associated protein of the cytoplasmic membrane of thefirst leukocyte. In another embodiment, the low-immunogenicbiocompatible polymer is a polyethylene glycol (PEG) (a methoxypolyethylene glycol (mPEG) for example). In still another embodiment,the process further comprises covalently binding the mPEG by contactingthe first leukocyte with methoxypoly(-ethylene glycol) succinimidylvalerate. In an embodiment, step (ii) occurs in vitro. In suchembodiment, the conditioned preparation can be a supernatant of a cellculture of the first leukocyte and the second leukocyte. In a furtherembodiment, the process further comprises preventing one of the firstleukocyte or the second leukocyte from proliferating prior to step (ii).In another embodiment, step (ii) occurs in vivo and comprisesadministering the first modified leukocyte to a mammal having/bearingthe second leukocyte. In such embodiment, the conditioned preparationcan be plasma. In yet another embodiment, the process further comprisespreventing the first leukocyte from proliferating prior toadministration to the mammal.

In an embodiment, step (iii) further comprises removing componentshaving an average molecular weight of more than about 10 kDa from theconditioned preparation or filtering out components having the averagemolecular weight of more than about 10 kDa from the conditionedpreparation. In still another embodiment, step (iii) further comprisesenriching the conditioned preparation in at least one miRNA species. Inyet another embodiment, step (iv) further comprises formulating thecomposition for intravenous administration to the subject. In anembodiment, the first leukocyte and/or the second leukocyte is a T cell(for example a CD4-positive T cell or a CD8-positive T cell).

According to a second aspect, the present invention provides apro-tolerogenic preparation obtained by the process described herein. Inan embodiment, the pro-tolerogenic preparation has at least one miRNAspecies presented in FIG. 13, at least one of the miRNA species listedany one of Tables 1A to 1D, at least one of the miRNA species listed inany one of Tables 2A to 2D and/or at least one of the miRNA speciesidentified in any one of FIGS. 12A to 12C.

According to a third aspect, the present invention provides a method ofincreasing a ratio of the level of regulatory T (Treg) cells to thelevel of pro-inflammatory T cells in a subject in need thereof. Broadly,the method comprises administering to the subject a therapeutic amountof a pro-tolerogenic preparation as described herein and/or obtained bythe process described herein so as to increase the ratio in the treatedsubject. In an embodiment, the increased ratio between the level of Tregcells and the level of pro-inflammatory T cells is for treating,preventing and/or alleviating the symptoms associated to an auto-immunedisease afflicting the subject. Auto-immune diseases include, but arenot limited to, type I diabetes, rheumatoid arthritis, multiplesclerosis, psoriasis, lupus, immune thrombocytopenia, experimentalautoimmune encephalomyelitis, autoimmune uveitis, inflammatory boweldisease, scleroderma and/or Crohn's disease. In another embodiment, theincreased ratio between the level of Treg cells and the level ofpro-inflammatory T cells is for preventing or limiting the rejection oftransplanted cells or tissue in the subject (for example, transplantedcells or tissue which are allogeneic or xenogeneic to the subject). Instill another embodiment, the increased ratio between the level of Tregcells and the level of pro-inflammatory T cells is for preventing orlimiting graft-versus-host disease in the treated subject. In anotherembodiment, the process for obtaining the pro-tolerogenic preparationcan further comprise formulating the composition for administrationprior to the transplantation of the cells or tissue.

According to a fourth aspect, the present invention provides apro-tolerogenic preparation for increasing a ratio of the level ofregulatory T (Treg) cells to the level of pro-inflammatory T cells in asubject. The present invention also provides a pro-tolerogenicpreparation for the preparation of a medicament for increasing a ratioof the level of regulatory T (Treg) cells to the level ofpro-inflammatory T cells in a subject. The pro-tolerogenic preparationis described herein and/or is obtained by the process described herein.In an embodiment, the pro-tolerogenic preparation is for treating,preventing and/or alleviating the symptoms associated to an auto-immunedisease afflicting the subject. Auto-immune diseases include, but arenot limited to, type I diabetes, rheumatoid arthritis, multiplesclerosis, psoriasis, lupus, immune thrombocytopenia, experimentalautoimmune encephalomyelitis, autoimmune uveitis, inflammatory boweldisease, scleroderma and/or Crohn's disease. In another embodiment, thepro-tolerogenic preparation is for preventing or limiting the rejectionof transplanted cells or tissue in the subject (for example,transplanted cells or tissue which are allogeneic or xenogeneic to thesubject). In still another embodiment, the pro-tolerogenic preparationis for preventing or limiting graft-versus-host disease in the treatedsubject. In another embodiment, the process for obtaining thepro-tolereogenic preparation can further comprise formulating thecomposition for administration prior to the transplantation of the cellsor tissue.

Throughout this text, various terms are used according to their plaindefinition in the art. However, for purposes of clarity, some specificterms are defined below.

Allogeneic cell. A cell is considered “allogeneic” with respect toanother cell if both cells are derived from the same animal species butpresents sequence variation in at least one genetic locus. A cell isconsidered “allogeneic” with respect to a subject if the cell is derivedfrom the same animal species as the subject but presents sequencevariation in at least one genetic locus when compared to the subject'srespective genetic locus. Allogeneic cells induce an immune reaction(such as a rejection) when they are introduced into an immunocompetenthost. In an embodiment, a first cell is considered allogeneic withrespect to a second cell if the first cell is HLA-disparate (orHLA-mismatched) with the second cell.

Allo-recognition. As it is known in the art, the term “allo-recognition”(also spelled allorecognition) refers to an immune response to foreignantigens (also referred to as alloantigens) from members of the samespecies and is caused by the difference between products of highlypolymorphic genes. Among the most highly polymorphic genes are thoseencoding the MHC complex which are most highly expressed on leukocytesthough other polymorphic proteins may similarly result in immunerecognition. These polymorphic products are typically recognized by Tcells and other mononuclear leukocytes. In the context of the presentinvention, the term “pro-inflammatory allo-recognition” refers to animmune response associated with the expansion of pro-inflammatory Tcells and/or the differentiation of naïve T cells into pro-inflammatoryT cells. Pro-inflammatory allo-recognition in vivo mediates cell ortissue injury and/or death and loss of cell or tissue function. Still inthe context of the present invention, the term “pro-tolerogenicallo-recognition” refers to an immune response associated with theexpansion of Treg cells and/or the differentiation of naïve T cells intoTreg cells. A pro-tolerogenic allo-recognition is usually consideredweaker than a pro-inflammatory allo-recognition. Further, an in vivopro-tolerogenic allo-recognition does not lead to significant cell ortissue injury and/or death nor loss of cell or tissue function.Anergy and Tolerance. In the present context, the term “anergy” refersto a non-specific state of immune unresponsiveness to an antigen towhich the host was previously sensitized to or unsensitized to. It canbe characterized by a decrease or even an absence of lymphokinesecretion by viable T cells when the T cell receptor is engaged by anantigen. In the present context, the term “tolerance” refers to anacquired specific failure of the immunological mechanism to respond to agiven antigen, induced by exposure to the antigen. Tolerance refers to aspecific non-reactivity of the immune system to a particular antigen,which is capable, under other conditions, of inducing an immuneresponse. However, in the present context, the terms “anergy” and“tolerance” are used interchangeably since the compositions and methodspresented herewith can be used to achieve both anergy and tolerance.

Autologous cell. A cell is considered “autologous” with respect toanother cell if both cells are derived from the same individual or fromgenetically identical twins. A cell is considered “autologous” to asubject, if the cell is derived from the subject or a geneticallyidentical twin. Autologous cells do not induce an immune reaction (suchas a rejection) when they are introduced into an immunocompetent host.

Immunogenic cell. A first cell is considered immunogenic with respect toa second cell when it is able to induce an immune response in the lattercell. In some embodiment, the immune response is in vitro (e.g. a mixedlymphocyte reaction) or can be observed in vivo (e.g. in a subjecthaving the second cell and having received the first cell). The secondcell can be located in an immunocompetent subject. Preferably, theimmune response is a cell-based immune response in which cellularmediator can be produced. In the context of this invention, theimmunogenic cells are immune cells, such as white blood cells orleukocytes.

Immunogenic cell culture conditions. A cell culture is considered to beconducted in immunogenic conditions when it allows the establishment ofa pro-inflammatory immune response between two distinct and unmodifiedleukocytes (and, in an embodiment, allo-recognition). Preferably, thepro-inflammatory immune response is a cell-based immune response inwhich cellular mediator can be produced. For example, the cell cultureconditions can be those of a mixed lymphocyte reaction (primary orsecondary). When a cell culture is conducted in immunogenic conditionsbut with leukocytes which have been modified to preventallo-recognition, no pro-inflammatory immune response is observed.However, when a cell culture is conducted in immunogenic conditions butwith leukocytes which have been modified to prevent pro-inflammatoryallo-recognition, a non-inflammatory or pro-tolerogenic immune responsecan be observed (for example a differentiation of naïve T cells to Tregcells and/or expansion of Treg cells).

Leukocyte. As used herein, a leukocyte (also spelled leucocyte) isdefined as a blood cell lacking hemoglobin and having a nucleus.Leukocytes are produced and derived from hematopoietic stem cells.Leukocytes are also referred to as white blood cells. Leukocytes includegranulocytes (also known as polymorphonuclear leucocytes), e.g.neutrophils, basophils and eosoniphils. Leukocytes also includeagranulocytes (or mononuclear leucocytes), e.g. lymphocytes, monocytesand macrophages. Some of the lymphocytes, referred to as T cells (orT-cell), bear on their surface a T-cell receptor. T cell are broadlydivided into cells expressing CD4 on their surface (also referred to asCD4-positive cells) and cells expressing CD8 on their surface (alsoreferred to as CD8-positive cells). Some of the lymphocytes, referred toas B cells (or B-cells), bear on their surface a B-cell receptor.

Low-immunogenic biocompatible polymer. As used herein, a“low-immunogenic polymer” refers to a polymer which is not or isunlikely to elicit an immune response in an individual. Thislow-immunogenic polymer is also capable of masking an antigenicdeterminant of a cell and lowering or even preventing an immune responseto the antigenic determinant when the antigenic determinant isintroduced into a subject. A “biocompatible polymer” refers to a polymerwhich is non-toxic when introduced into a subject. Exemplarylow-immunogenic biocompatible polymers includes, but are not limited to,polyethylene glycol (for example methoxypoly(ethylene glycol)),hyperbranched polyglycerol (HPG) and 2-alkyloxazoline (POZ).

Non-proliferative leukocyte. As used herein, the term “non-proliferativeleukocyte” refers to a lymphocyte which has been modified to no longerbeing capable of cellular proliferation (e.g. performing at least onecomplete division cycle). In some embodiments, this modification may betemporary and the non-proliferative properties of a leukocyte may belimited in time. For example, when a leukocyte is modified from acontact with a pharmacological agent capable of limiting itsproliferation, the removal of the pharmacological agent from the cellculture can allow the leukocyte to regain its proliferative properties.In other embodiments, the modification is permanent and the modifiedleukocyte cannot regain its proliferative properties. For example, whena leukocyte is irradiated, it is not possible for it to regain itsproliferative properties. In the context of the present application, theexpressions “non-proliferative leukocyte” or “leukocyte limited fromproliferating” are used interchangeably.

Peripheral blood mononuclear cells (PBMC). This term refers to the cellpopulation recuperated/derived from the peripheral blood of a subject(usually a mammal such as a human). PBMC usually contains T cells, Bcells and antigen presenting cells.

Pharmaceutically effective amount or therapeutically effective amount.These expressions refer to an amount (dose) of an acellular preparationeffective in mediating a therapeutic benefit to a patient (for exampleprevention, treatment and/or alleviation of symptoms of animmune-associated disorder in which the ratio of Tregs topro-inflammatory T cells is low when compared to sex- and age-matchedhealthy subjects). It is also to be understood herein that a“pharmaceutically effective amount” may be interpreted as an amountgiving a desired therapeutic effect, either taken in one dose or in anydosage or route, taken alone or in combination with other therapeuticagents.

Prevention, treatment and alleviation of symptoms. These expressionsrefer to the ability of the acellular preparation to limit thedevelopment, progression and/or symptomology of a immune-associateddisorder associated to an abnormal/excessive immune response (forexample prevention, treatment and/or alleviation of symptoms of animmune-associated disorder in which the ratio of Tregs topro-inflammatory T cells is low when compared to sex- and age-matchedhealthy subject). Broadly, the prevention, treatment and/or alleviationof symptoms encompasses increasing the levels of Treg cells and/ordecreasing the levels of pro-inflammatory T cells. The acellularpreparation is considered effective or successful for treating and/oralleviating the symptoms associated with the disorder when a reductionin the pro-inflammatory state (when compared to an untreated andafflicted individual) in the treated individual (previously known to beafflicted with the disorder) is observed. The acellular-basedpreparation is considered effective or successful for preventing thedisorder when a reduction in the pro-inflammatory state (when comparedto an untreated and afflicted individual) in the treated individual isobserved upon an immunological challenge (such as, for example, anantigenic challenge).

Pro-inflammatory T cells. In the present context, pro-inflammatory Tcells are a population of T cells capable of mediating an inflammatoryreaction. Pro-inflammatory T cells generally include T helper 1 (Th1 orType 1) and T helper 17 (Th17) subsets of T cells. Th1 cells partnermainly with macrophage and can produce interferon-γ, tumor necrosisfactor-β, IL-2 and IL-10. Th1 cells promote the cellular immune responseby maximizing the killing efficacy of the macrophages and theproliferation of cytotoxic CD8⁺ T cells. Th1 cells can also promote theproduction of opsonizing antibodies. T helper 17 cells (Th17) are asubset of T helper cells capable of producing interleukin 17 (IL-17) andare thought to play a key role in autoimmune diseases and in microbialinfections. Th17 cells primarily produce two main members of the IL-17family, IL-17A and IL-17F, which are involved in the recruitment,activation and migration of neutrophils. Th17 cells also secrete IL-21and IL-22.

Regulatory T cells. Regulatory T cells are also referred to as Treg andwere formerly known as suppressor T cell. Regulatory T cells are acomponent of the immune system that suppress immune responses of othercells. Regulatory T cells usually express CD3, CD4, CD8, CD25, andFoxp3. Additional regulatory T cell populations include Tr1, Th3,CD8⁺CD28⁻, CD69⁺, and Qa-1 restricted T cells. Regulatory T cellsactively suppress activation of the immune system and preventpathological self-reactivity, i.e. autoimmune disease. The critical roleregulatory T cells play within the immune system is evidenced by thesevere autoimmune syndrome that results from a genetic deficiency inregulatory T cells. The immunosuppressive cytokines TGF-β andInterleukin 10 (IL-10) have also been implicated in regulatory T cellfunction. Similar to other T cells, a subset of regulatory T cells candevelop in the thymus and this subset is usually referred to as naturalTreg (or nTreg). Another type of regulatory T cell (induced Treg oriTreg) can develop in the periphery from naïve CD4⁺ T cells. The largemajority of Foxp3-expressing regulatory T cells are found within themajor histocompatibility complex (MHC) class II restrictedCD4-expressing (CD4⁺) helper T cell population and express high levelsof the interleukin-2 receptor alpha chain (CD25). In addition to theFoxp3-expressing CD4⁺CD25⁺, there also appears to be a minor populationof MHC class I restricted CD8⁺ Foxp3-expressing regulatory T cells.Unlike conventional T cells, regulatory T cells do not produce IL-2 andare therefore anergic at baseline. An alternative way of identifyingregulatory T cells is to determine the DNA methylation pattern of aportion of the foxp3 gene (TSDR, Treg-specific-demethylated region)which is found demethylated in Tregs.

Splenocytes. This term refers to the cell population obtained from thespleen of a subject (usually a mammal such as a rodent). Splenocytesusually comprise T cell, B cell as well as antigen presenting cells.

Syngeneic cell. A cell is considered “syngeneic” with respect to asubject (or a cell derived therefrom) if it is sufficiently identical tothe subject so as to prevent an immune rejection upon transplantation.Syngeneic cells are derived from the same animal species.

Viable. In the present context, the term “viable” refers to the abilityof a cell to complete at least one cell cycle and, ultimatelyproliferate. A viable cell is thus capable of proliferating. Byopposition, the term “non-viable” or “non-proliferative” both refer to acell which is no longer capable of completing at least one cell cycle.By comparison, the term “cycle arrest” refers to a cell which has beentreated to halt its cell cycle progression (usually with apharmacological agent) but which is still capable of re-entering thecell cycle (usually when the pharmacological agent is removed).

Xenogeneic cell. A cell is considered “xenogeneic” with respect to asubject (or a cell derived from the subject) when it is derived from adifferent animal species than the subject. A xenogeneic cell is expectedto be rejected when transplanted in an immunocompetent host.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof.

FIG. 1 illustrates molecular weight fractionation studies of theacellular preparations demonstrate that the majority of immunomodulatoryactivity (denoted by *) resides in the low (<10 kDa) molecular weightfraction. Results are shown as percent Treg lymphocytes in function ofculture conditions (resting PBMC, MLR or mPEG-MLR) as well as weightfractionation (unfractionated media; >10 kDa fraction; <10 kDa fractionas indicated on the legend). Dashed line represents baseline levels.

FIG. 2 illustrates the effects of size (MW) separation and RNasetreatment on the immunomodulary effects of acellular preparations.Unmodified conditioned murine plasma (obtained from donor mice 5 dayspost splenocyte transfer), size fractionated-conditioned murine plasmaor RNase-treated conditioned murine plasma was administered once tonaïve mice and Treg/Th17 levels were measured (when) in the spleen. (A)Results are shown as the percentage of Treg cells (in function of CD4⁺cells) in function of type of conditioned medium (white bars=conditionedplasma obtained from administering saline, hatched bars=conditionedplasma obtained from administering unmodified allogeneic splenocytes,grey bars=conditioned plasma obtained from administering polymermodified allogeneic splenocytes) and size fractionation (non-fractionedor complete conditioned serum, fraction>100 kDa, fraction between 30 and100 kDa, fraction between 10 and 30 kDa, fraction<10 kDa). a denotes themean value for unfractionated conditioned medium prepared from micepreviously treated with unmodified allogeneic cells. b denotes the meanvalue for unfractionated conditioned medium prepared from micepreviously treated with mPEG-modified allogeneic cells. (B) Results areshown as the percentage of Th17 cells (in function of CD4⁺ cells) infunction of type of conditioned medium (white bars=conditioned plasmaobtained from administering saline, hatched bars=conditioned plasmaobtained from administering unmodified allogeneic splenocytes, greybars=conditioned plasma obtained from administering polymer modifiedallogeneic splenocytes) and size fractionation (non-fractioned orcomplete conditioned serum, fraction>100 kDa, fraction between 30 and100 kDa, fraction between 10 and 30 kDa, fraction<10 kDa). a denotes themean value for unfractionated conditioned medium prepared from micepreviously treated with unmodified allogeneic cells. b denotes the meanvalue for unfractionated conditioned medium prepared from micepreviously treated with mPEG-modified allogeneic cells. (C) Results areshown as the percentage of Treg cells (in function of CD4⁺ cells, leftpanel) or Th17 cells (in function of CD4⁺ cells, right panel) infunction of type of treatment (white bars=N=naïve untreated animals;grey bars=AC=unmodified allogeneic cells; diagonal hatchbars=conditioned plasma obtained from administered unmodifiedsplenocytes treated (allo-plasma (+)) or not (allo-plasma (−)) withRNase; horizontal hatch bars=conditioned plasma obtained fromadministering polymer modified splenocytes treated (mPEG-allo-plasma(+)) or not (mPEG-allo-plasma (−)) with RNase).

FIG. 3 illustrates the size fractionated conditioned plasma on theintracellular expression of cytokines. Unmodified conditioned murineplasma (obtained from donor mice 5 days post saline or splenocytetransfer), size fractionated-conditioned murine plasma was administeredonce to naïve mice and Treg/Th17 levels were measured (when) in thespleen. Results are shown as the percentage intracellular cytokinepositive CD4⁺ cells in function of type of conditioned medium (whitebars=conditioned plasma obtained from administering saline, hatchedbars=conditioned plasma obtained from administering unmodifiedallogeneic splenocytes, grey bars=conditioned plasma obtained fromadministering polymer modified allogeneic splenocytes) and sizefractionation (non-fractioned or complete conditioned serum,fraction>100 kDa, fraction between 30 and 100 kDa, fraction between 10and 30 kDa, fraction<10 kDa) for (A) IL-10, (B) IL-2, (C) IFN-γ, (D)TNF-α and (E) IL-4. * denotes p<0.001 relative to treatment withconditioned plasma from mice treated with saline, # denotes p<0.001relative to treatment with conditioned medium derived from mice treatedwith unmodified allogeneic splenocytes.

FIG. 4 illustrates the in vivo effects of the various conditioned mediumand preparations derived therefrom on the intracellular expression ofcytokines as well as type of CD4⁺ cells. Conditioned plasma was obtainedby administering naïve mice with saline, unmmodified allogeneicsplenocytes or polymer-modified allogeneic splenocytes (PEG) andrecuperating plasma after 5 days. The obtained plasma was eitheradministered directly (●=untreated) or optionally treated with RNaseA(◯=conditioned plasma, ▪=miRNA enriched fraction of conditioned plasma)and/or further purified so as to retain and enrich the <10 kDa fraction(e.g. miRNA) (▪=untreated miRNA, □=RNase A-treated miRNA) prior toadministration. As a control, RNase A was also administered directly tosome animals. After 30, 60, 120, 180, 270 days, animals were sacrificed,their spleen was removed and CD4⁺ cells were characterized by flowcytometry. Results are shown for intracellular cytokine expression: IL-2(A), INF-γ (B), IL-10 (C), as well as CD4⁺ cell type: Treg (Foxp3⁺) (D)and Th17 (IL-17⁺) (E) CD4⁺ cells.

FIG. 5 illustrates the effects of the TA1 preparations on thephosphorylation of phosphokinases of resting Jurkat cells. Results areshown as fold modulation (when compared to saline-treated Jurkat cells)for each kinase tested. (A) On this panel, Akt is considered to besignificantly increasingly phosphorylated. (B) On this panel, PRAS40 isconsidered to be significantly increasingly phosphorylated. (C) On thispanel, HSP60 is considered to be significantly decreasinglyphosphorylated. * denotes greater than 10-fold increase in proteinphosphorylation over resting Jurkat cells. # denotes greater than10-fold decrease in protein phosphorylation over resting Jurkat cells.

FIG. 6 shows the in vitro effects of the murine TA1 preparation on humanPBMC PBMCs. Murine TA1 preparation (either 25 μL, 50 μL, 100 μL or 200μL) was included in a human PBMC MLR assay and cellular proliferationwas measured. Results are shown as percent in proliferation (CD3⁺CD4⁺cells) in function of conditions (Rest=resting MLR, MLR=conventional MLRwithout TA1, Murine TA-1=MLR with TA1) and TA1 concentration (in μL)after 10 days (A) or 14 days (B). * denotes p<0.001 relative to MLRvalue and ¢ denotes the concentration of the TA1 preparation used in thein vivo mouse study.

FIG. 7 compares the in vitro immunosuppressive effects of TA1preparation and etanercept. A mixed lymphocyte reaction using Balb/c andC57Bl/6 splenocytes was conducted in the absence (▪=control MLR,□=control MLR with sham TA1) or the presence of etanercept (◯, x axisprovides concentration in ng/mL used) or the TA1 composition (●, x axisprovides concentration in μL/mL used). (A) Results are shown as thepercentage of proliferation of CD4⁺ splenocytes in function of treatmentafter 8 days (solid line) and 14 days (dashed line). (B) Results areshown as the percentage of proliferation of CD8⁺ splenocytes in functionof treatment after 8 days (solid line) and 14 days (dashed line).

FIG. 8 illustrates significant changes in the levels of Th17 and Treglymphocytes are noted in the spleen (upper panels), brachial lymph node(middle panels) and pancreatic lymph nodes (lower panels) uponconversion of NOD mice from non-diabetic (left panels) to diabetic(right panels). These changes are characterized by dramaticallyincreased Th17 (in the spleen, from 0.03 to 3.84%; in the brachial lymphnode, from 0.01% to 0.67%; in the pancreatic lymph node, from 0.05% to1.05%) and significantly decreased Treg (in the spleen, from 16.5% to2.0%; in the brachial lymph node from 11.8% to 1.8% and in thepancreatic lymph node, from 12.7% to 4.1%) lymphocytes. Tregs: *,p<0.001 from non-diabetic NOD mice. Th17: ** p<0.001 from non-diabeticNOD mice.

FIG. 9 illustrates the effects of the TA1 preparations in NOD mice. TA1acellular preparation was manufactured from mice treated withmPEG-modified allogeneic splenocytes five (5) days post treatment. Thepurified miRNA composition was administered thrice (100 μl per i.v.injection at days 0, 2 and 4) to 7 week-old NOD mice (n=15). Controlmice (n=16; untreated) were injected with 100 μl of saline. (A) Theglycaemia of the animals were determined during the weeks following thetreatment. Results are shown as the percentage of normoglycemic animalsin function of age (in weeks) and treatment (dashed line=TA1-treated,solid line=naïve untreated animals). In this model, diabetes begins tooccur at approximately 15 weeks. Between weeks 15 and 20, 75% ofuntreated mice developed hyperglycemia (i.e. diabetes) compared to 13%of TA1-treated mice. After 30 weeks, 9 out of the 15 animals treatedwith TA1 were still considered normoglycemic compared to only 4 out ofthe 16 for untreated animals. In TA1-treated mice developing diabetes (6out of 15), 67% of the animals exhibited delayed onset (post 20 weeks)relative to untreated mice where 100% of the diabetic mice arose priorto 20 weeks of age. (B) The Treg/Th17 ratio in the pancreatic lymph nodewas determined during the weeks following treatment. Results are shownas the log in Treg/Th17 ratio in function of age (weeks) in micedeveloping diabetes (open circles/dashed line=untreated mice; closedcircles/solid=TA1-treated mice). At 30 weeks of age, all surviving micewere sacrificed and the Treg/Th17 ration was determined (openstar=untreated mice±range; closed star=TA1-treated mice±range). (C) TheTreg/Th17 ratio in the pancreatic lymph note was determined in healthy20-week old Balb/c (198) and C57Bl/6 (91.5), 7-week old NOD (103),diabetic NOD (control animals (4.7) and TA1-treated (70) animals) aswell as 30-week old non-diabetic NOD mice (control animals (286) andTA1-treated animals (255).

FIG. 10 illustrates the in vivo effects of TA1 preparations ontolerogenic/anergic immune cell populations. NOD mice (7 week-old) weretreated trice (100 μL i.v. at days 0, 2 and 4) with either saline or themurine TA1 preparations. As mice became diabetic or at 30 weeks of age,immune populations were characterized in the spleen, the brachial lymphnode, the pancreatic lymph node and, for Treg cells, the thymus. In thisfigure, light grey bars refer to saline-treated animals and dark greybars refers to TA1-treated animals. (A) Results are shown as thepercentage of Foxp3⁺ (Treg) cells (in function of total CD4⁺ cells) infunction of treatment. (B) Results are shown as the percentage of TGF-β⁺(Treg and Th2) cells (in function of total CD4⁺ cells) in function oftreatment. (C) Results are shown as the percentage of IL-4⁺ (Th2 andnaive) cells (in function of total CD4⁺ cells) in function of treatment.(D) Results are shown as the percentage of IL-10⁺ (Treg and Th2) cells(in function of total CD4⁺ cells) in function of treatment. (E) Resultsare shown as the percentage of CD62L⁺ (Treg) cells (in function of totalCD4⁺ cells) in function of treatment. (F) Results are shown as thepercentage of CD152⁺ cells (in function of total CD4⁺ cells) in functionof treatment. (G) Results are shown as the percentage of CD11c⁺ cells(in function of total DC cells) in function of treatment. * denotesp<0.001 relative to saline-treated animals.

FIG. 11 illustrates the in vivo effects of TA1 preparations onpro-inflammatory immune cell populations. NOD mice (7 week-old) weretreated trice (100 μL i.v. at days 0, 2 and 4) with either saline orTA1. As mice became diabetic (weeks 15-29) or at 30 weeks of age weresacrificed and immune populations were characterized in the spleen, thebrachial lymph node, the pancreatic lymph node and, for Th17 cells, thethymus. In this figure, light grey bars refer to saline-treated animalsand dark grey bars refers to TA1-treated animals. (A) Results are shownas the percentage of IL-17A⁺ (Th17) cells (in function of total CD4⁺cells) in function of treatment. (B) Results are shown as the percentageof INF-γ⁺ (Th1) cells (in function of total CD4⁺ cells) in function oftreatment. (C) Results are shown as the percentage of IL-2⁺ (Th1) cells(in function of total CD4⁺ cells) in function of treatment. (D) Resultsare shown as the percentage of TNF-α⁺ (Th1) cells (in function of totalCD4⁺ cells) in function of treatment. (E) Results are shown as thepercentage of IL-12⁺ (Th1) cells (in function of total CD4⁺ cells) infunction of treatment. (F) Results are shown as the percentage of NK1.1⁺cells (in function of total TCR-α/β+ cells) in function of treatment. *denotes p<0.001 relative to saline treated animals.

FIG. 12 provides a comparison of the miRNA populations between differentMLR assays. A human PBMC MLR assay (using unmodified (control MLR) orpolymer modified leukocyte (mPEG MLR)) was conducted and miRNA contentwas partially determined. Volcano plots of comparing the miRNApopulation of the conditioned medium of the control MLR to the one ofthe supernatant of resting cells (A), comparing the miRNA population ofthe conditioned medium of a mPEG MLR to the one of the supernatant ofresting cells (B) and comparing the miRNA population of the conditionedmedium of a mPEG MLR to the one of the conditioned medium of a controlMLR(C) are provided. Results are provided in −Log₁₀ (p value) infunction of Log₂ fold change. In these volcano plots, the followingmiRNAs have been identified with numbers:

-   -   1 has-miR-298    -   2 has-miR-34a-5p    -   3 has-miR-574-3p    -   4 has-miR-125b-5p    -   5 has-let-7a-5p    -   6 has-miR-196a-5p    -   7 has-miR-148a-3p    -   8 has-let-7e-5p    -   9 has-miR-134

FIG. 13 provides a partial miRNA compositional analysis of theconditioned medium of a mPEG MLR (white bars) and of a control MLR(black bars). Results are provided, for each miRNA, as log₂ foldregulation when compared to the miRNA present in the supernatant ofresting cells. White open stars denote Log₂-fold change and black solidstars denote significant changes in volcano plot analysis.

FIG. 14 provides a selection of the miRNA compositional analysis of theconditioned medium of a mPEG MLR (white bars) and of a control MLR(black bars). Results are provided, for each miRNA, as log₂ foldregulation when compared to the miRNA present in the supernatant ofresting cells. White open stars denote Log₂-fold change and black solidstars denote significant changes and or clustergram (heatmap) determinedmiRNA shifts denoted in volcano plot analysis.

DETAILED DESCRIPTION

In accordance with the present invention, there are provided acellularpreparations obtained by contacting two distinct allogeneic leukocytepopulations wherein at least one of the two leukocyte populations ismodified to bear on its surface a low-immunogenic biocompatible polymer.The two leukocyte populations are contacted under conditions so as toallow pro-tolerogenic allo-recognition (e.g. expanding of Treg cellsand/or differentiation of naïve cells into T reg cells) and limitpro-inflammatory allo-recognition. The acellular components produced bycontacting the two leukocyte populations can optionally be purified orenriched to provide a preparation enriched in miRNAs. In embodiments,the acellular preparation can also be processed to (substantially)remove cells, cells fragments as well as secreted proteins (such ascytokines for example). The contact between the two leukocytepopulations can occur in vitro, ex vivo or in vivo.

These acellular preparations induce a state of (complete or partial)immune tolerance, immuno-quiescence or anergy. As such these acellularpreparations can be useful for increasing the levels of regulatory Tcells and/or decreasing the levels of pro-inflammatory T cells insubjects in need thereof.

The acellular preparations can be obtained by modifying a firstleukocyte to bear on its surface a low-immunogenic biocompatible polymerand contacting the first leukocyte with a second leukocyte (consideredallogeneic with respect to the first leukocyte). The contact can be madein vitro by co-culturing the first leukocyte and the second leukocyteunder conditions so as to allow pro-tolerogenic allo-recognition andlimit (or inhibit) pro-inflammatory allo-reocgnition. In suchembodiment, the cell culture (or a portion thereof such as thesupernatant of the cell culture) is recuperated and processed tosubstantially remove the cells it may contain to provide the acellularpreparation. Alternatively, the contact can be made in vivo byintroducing the first leukocyte in a test subject bearing the secondleukocyte (such as a non-human animal or mammal). The first leukocyte isallogeneic or xenogeneic to the test subject. In such embodiment, theblood or a blood fraction (such as serum or plasma) is recuperated fromthe test subject and processed to substantially remove the cells it maycontain to provide the acellular preparation.

Since the acellular preparations can optionally be enriched in miRNAs,it is important that the cell culture and/or the blood/blood fraction beprocessed in conditions so as to retain the integrity of the majority ofthe miRNA species present, for example by substantially inhibiting RNAdegradation. As used herein, the term “substantially inhibiting RNAdegradation” indicate that the conditions allow for the degradation ofless than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6% or 5% of the miRNA population obtained by RNases. RNases include,but are not limited endoribonucleases (e.g., RNase A, RNase H, RNase I,RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2,RNase V1 and/or RNase V) and exoribonucleases (e.g. polynucleotidepPhosphorylase (PNPase), RNase PH, RNase II, RNase R, RNase D, RNase T,Oligoribonuclease, Exoribonuclease I and/or Exoribonuclease II). Sinceit is known in the art that miRNAs are, in general, more resistancetowards degradation than messenger RNAs, the conditions for obtainingand processing the cell culture/blood can allow for some RNAdegradation, preferably limited to the mRNA fraction.

As it will be shown below, acellular preparations obtained frompolymer-based bioengineering of allogeneic leukocytic cells provides asignificant opportunity to modulate the responsiveness (i.e.,immunoquiescent versus pro-inflammatory) of the recipient's immunesystem. Without wishing to be bound to theory, it is hypothesized thatthe acellular preparations obtained from using polymer-modifiedleukocytes can be used to induce Tregs differentiation/expansion and/orattenuate Th17/1 and pro-inflammatory cytokine upregulation to preventor lower a pro-inflammatory immune response. Moreover, specific NK(natural killer) cell (NK1.1) upregulation is favored in tissuesexhibiting autoimmune damage. NK1.1 positive cells are reported to beimportant in the killing of self-reactive immune cells. Moreover, it isproposed that the acellular preparations obtained from usingpolymer-modified allogeneic leukocytes can be used therapeutically invarious diseases (such as auto-immunity or an excessive immune response)to increase the levels Treg cells and/or decrease pro-inflammatoryeffector cells, to ultimately increase the ratio of regulatory T cellsto pro-inflammatory T cells thereby attenuating the incidence and/orseverity of the disease pathology.

Processes for Obtaining Acellular Preparations

The acellular preparations presented described herein can be obtained bycontacting two distinct and allogeneic leukocyte populations (referredherein to the first leukocyte and the second leukocyte). The firstleukocyte is modified to bear on its surface (and, in some embodiment,to be covalently bound to) a low-immunogenic biocompatible polymer.Optionally, the second leukocyte can also be modified to bear on itssurface (and, in some embodiment, to be covalently bound to) alow-immunogenic biocompatible polymer. The two leukocyte populations arecontacted under conditions so as to limit (and in some embodimentsprevent) pro-inflammatory allo-recognition and to allow pro-tolerogenicallo-recognition.

It is important that the polymer used exhibits both low-immunogenicityand biocompatibility once introduced into a cell culture system oradministered to the test subject. Polyethylene glycol (particularlymethoxypoly(ethylene glycol)), 2-alkyloxazoline (POZ) and hyperbranchedpolyglycerol (HPG) are exemplary polymers which all exhibit lowimmunogenicity and biocompatibility and can be successfully used tomodify the first leukocyte (and optionally the second leukocyte). Insome embodiments, it is preferable to use a single type of polymer tomodify the surface of leukocytes. In other embodiments, it is possibleto use at least two distinct types of polymers to modify the surface ofthe leukocyte.

In an embodiment, the low-immunogenic biocompatible polymer can becovalently associated with the membrane-associated protein(s) of theleukocyte by creating a reactive site on the polymer (for example bydeprotecting a chemical group) and contacting the polymer with theleukocyte. For example, for covalently binding a methoxypoly(ethyleneglycol) to the surface of a leukocyte, it is possible to incubate amethoxypoly(-ethylene glycol) succinimidyl valerate (reactive polymer)in the presence of the leukocyte. The contact between the reactivepolymer and the leukocyte is performed under conditions sufficient forproviding a grafting density which will limit/prevent pro-inflammatoryallo-recognition and allow pro-tolerogenic allo-recognition. In anembodiment, the polymer is grafted to a viable leukocyte and underconditions which will retain the viability of the leukocyte. A linker,positioned between the surface of the leukocyte and the polymer, canoptionally be used. Examples of such polymers and linkers are describedin U.S. Pat. Nos. 5,908,624; 8,007,784 and 8,067,151. In anotherembodiment, the low-immunogenic biocompatible polymer can be integratedwithin the lipid bilayer of the cytoplasmic membrane of the leukocyte byusing a lipid-modified polymer.

As indicated above, it is important that the low-immunogenicbiocompatible polymer be grafted at a density sufficient forlimiting/preventing pro-inflammatory allo-recognition while allowingpro-tolerogenic allo-recognition of the first leukocyte by the secondleukocyte. In an embodiment, the polymer is polyethylene glycol (e.g.linear) and has an average molecular weight between 2 and 40 KDa as wellas any combinations thereof. In a further embodiment, the averagemolecular weight of the PEG to be grafted is at least 2, 3, 4, 5, 10,15, 20, 25, 30, 35 or 40 kDa. In another embodiment, the averagemolecular weight of the PEG to be granted is no more than 40, 35, 30,25, 20, 15, 10, 5, 4, 3, or 2 kDa. In still another embodiment, thegrafting concentration of the polymer (per 20×10⁶ cells) is between 1and 10 mM (preferably between 2.5 to 10 mM). In another embodiment, thegrafting concentration of the polymer (per 20×10⁶ cells) is at least 1,1.5, 2, 2.5, 5, 6, 7, 8, 9 or 10 mM. In still another embodiment, thegrafting concentration of the polymer (per 20×10⁶ cells) is no more than10, 9, 8, 7, 6, 5, 2.5, 2, 1.5 or 1 mM. In order to determine ifpro-inflammatory allo-recognition occurs (or is prevented), varioustechniques are known to those skilled in the art and include, but arenot limited to, a standard mixed lymphocyte reaction (MLR), highmolecular weight mitogen stimulation (e.g. PHA stimulation) as well asflow cytometry (Chen and Scott, 2006). In order to determine if apro-tolerogenic allo-recognition occurs (or is prevented), varioustechniques are known to those skilled in the art and include, but arenot limited to, the assessment of the level of expansion anddifferentiation of Treg cells and or prevention of Th17expansion/differentiation. In an embodiment, the polymer is selected andgrafted to the modified leukocyte to provide a modified leukocyte havinga pro-inflammatory/pro-tolerogenic allo-recognition substantiallysimilar to the one observed in a mixed lymphocyte reaction between afirst leukocyte modified to be grafted with 20 kDa mPEG at a density ofat least 0.5 mM (and preferably 1 mM, even more preferably 2.5 mM) per20×10⁶ cells and incubated with a second (unmodified) allogeneicleukocyte.

Before or after being modified with a low-immunogenic and biocompatiblepolymer, the first leukocyte can be optionally modified to refrain frombeing proliferative. This modification preferably occurs prior to itsintroduction in a cell culture system or its administration into a testsubject. For example, the leukocyte can be irradiated (e.g.γ-irradiation) prior to its introduction in a cell culture system or inthe test subject. Upon irradiation, the leukocyte is not consideredviable (e.g. capable of proliferation). In an embodiment, polymergrafting can affect the leukocyte viability and be used to refrain theleukocyte from proliferating. Alternatively, leukocyte can be treatedwith a pharmacological agent which halts cell cycle progression. Uponthe administration of such pharmacological agent, the leukocyte isconsidered viable since it can resume cellular proliferation when theagent is removed from the cell-containing medium.

It is also contemplated that the second leukocyte (which can optionallybe modified with the low-immunogenic and biocompatible polymer) be alsooptionally modified to refrain from being proliferative. For example,the leukocyte can be irradiated (e.g. γ-irradiation) prior to itsintroduction in a cell culture system or in the test subject. Uponirradiation, the leukocyte is not considered viable (e.g. capable ofproliferation). In an embodiment, polymer grafting can affect theleukocyte viability and can be used to refrain the leukocyte fromproliferating. Alternatively, leukocyte can be treated with apharmacological agent which halts cell cycle progression. Upon theadministration of such pharmacological agent, the leukocyte isconsidered viable since it can resume cellular proliferation when theagent is removed from the cell-containing medium. However, when thesecond leukocyte is modified from being proliferative, it is importantthe first leukocyte with which it is being contacted remainsproliferative.

In order to generate the acellular preparation, it is not necessary toprovide homogeneous leukocyte populations. For example, the firstleukocyte population (such as, for example a PBMCs or splenocytes) canbe introduced in a cell culture system and contacted with a secondleukocyte population (such as, for example a PBMCs or splenocytes) oradministered to the test subject. However, in some embodiments, it ispossible to provide and contact a more homogeneous leukocytepopulations. For example, the first leukocyte population can berelatively homogenous (such as, for example, a T cell population) andintroduced in a cell culture system comprising a second leukocyte (suchas, for example a PBMC or splenocyte) or administered to the testsubject. In another example, the first leukocyte population (such as,for example a PBMC or splenocyte) can be introduced in a cell culturesystem comprising a second leukocyte population which can be relativelyhomogeneous (such as, for example, a T cell population). In a furtherexample, the first leukocyte population can be relatively homogenous(such as, for example, a T cell population) and introduced in a cellculture system comprising a second leukocyte population which can berelatively homogeneous (such as, for example, a T cell population).

To provide the acellular preparations described herewith, the leukocytesused can be mature leukocytes or be provided in the form of stem cells.For example, leukocytes can be obtained from isolating peripheral bloodmononuclear cells (PBMC) from the subject. Optionally, the PBMCs can bedifferentiated in vitro into dendritic (DC) or DC-like cells.Alternatively, the leukocytes can be obtained from the spleen (e.g.splenocytes). Leukocytes usually include T cells, B cells and antigenpresenting cells. For providing the acellular preparations, theleukocytes are not erythrocytes since the polymer-modified erythrocytesare not capable of eliciting a pro-tolerogenic allo-recognition whenadministered in a test subject. However, traces of erythrocytes in theleukocyte population used are tolerated (for example, less than about10%, less than about 5% or less than about 1% of the total number ofcells in the preparation).

Even though it is not necessary to further purify the leukocytes toprovide the acellular preparations, it is possible to use a pure cellpopulation or a relatively homogenous population of cells as leukocytes.This “pure” cell population and “relative homogenous population” ofcells can, for example, essentially consist essentially of a single celltype of T cells, B cells, antigen presenting cells (APC) or stem cells.Alternatively, the population of cells can consist essentially of morethan one cell type. The population of cells can be obtained throughconventional methods (for example cell sorting or magnetic beads). In anembodiment, when the population of cells consist of a single cell type(for example, T cells), the percentage of the cell type with respect tothe total population of cells is at least 90%, at least 95% or at least99%. The relatively homogenous population of cells are expected tocontain some contaminating cells, for example less than 10%, less than5% or less than 1% of the total population of cells.

The first leukocyte and/or second leukocyte can be obtained from anyanimals, but are preferably derived from mammals (such as, for example,humans and mice). In an embodiment, the first and/or second leukocytecan be obtained from a subject intended to be treated with the acellularpreparation.

The first and/or second leukocyte can be expanded in vitro prior to theintroduction in a cell culture system or the administration to a testsubject.

As indicated above, the first and the second leukocyte are contactedunder conditions to limit/prevent pro-inflammatory allo-recognition(e.g. expansion of pro-inflammatory T cells and/or differentiation ofnaïve T cells in pro-inflammatory T cells) and allow pro-tolerogenicallo-recognition (e.g. expansion of Treg cells and/or differentiation ofnaïve T cells in Treg cells). When the contact occurs in vitro, it isimportant that the first leukocyte and the second leukocyte be culturedunder conditions allowing physical contact between the two leukocytepopulations and for a time sufficient to provide a conditioned medium.As used herein, a conditioned medium refers to physical components of acell culture (or fraction thereof, such as the cell culture supernatant)obtained by contacting the first and the second leukocyte and having thepro-tolerogenic properties described herein. Usually, the conditionedmedium is obtained at least 24 hours after the initial contact betweenthe first and the second leukocyte. In some embodiment, the conditionedmedium is obtained at least 48 hours or at least 72 hours after theinitial contact between the first and the second leukocyte. In anembodiment, the conditioned medium can be obtained after at least 24hours of incubating a first leukocyte (for example grafted with a 20 kDaPEG at a density of at 1.0 mM) with a second leukocyte. When theincubation takes place in a 24-well plate, the concentration of eachleukocyte population can be at least 1×10⁶ cells.

When the contact occurs in vivo, it is important that the firstleukocyte be administered to an immune competent test subject (bearingthe second leukocyte) and that the blood or blood fraction be obtainedat a later a time sufficient to provide a conditioned blood. The testsubject is a subject being immune competent and having aTreg/pro-inflammatory ratio which is substantially similar to age- andsex-matched healthy subjects. As used herein, the conditioned bloodrefers to physical components present in the blood (or fraction thereof,such as the plasma) obtained by administering the first leukocyte to theimmune competent test subject and having the pro-tolerogenic propertiesdescribed herein. It is recognized by those skilled in the art that theconditioned blood may be obtained more rapidly by increasing the amountof leukocytes being administered or administering more than once (forexample one, twice or thrice) the polymer-modified leukocyte. Usually,the conditioned blood is obtained at least one day after theadministration of the first leukocyte. In some embodiment, theconditioned blood is obtained at least 2, 3, 4, 5, 6 or 7 days after theadministration of the first leukocyte. In an embodiment, the conditionedblood can be obtained by administering at least 5×10⁶ polymer-modifiedleukocytes (for example grafted with at least 1.0 mM of 20 kDa PEG) tothe test subject (e.g. a mice) and recuperating the plasma five dayslater. In some embodiments, the conditioned blood can be obtained byadministering at least 20×10⁶ polymer-modified leukocytes.

As indicated herein, the two leukocyte populations are consideredallogeneic (and in some embodiments, xenogeneic). When the acellularpreparation is obtained in vivo by, for example, obtaining a conditionedblood/blood fraction by administering the first leukocyte to the testsubject, the first leukocyte can be allogeneic or xenogeneic to the testsubject. In such embodiment, it is also contemplated that the firstleukocyte be autologous, syngeneic, allogeneic or xenogeneic to atreated subject who is going to receive the acellular preparation. Whenthe acellular preparation is obtained in vitro by, for example,obtaining a conditioned medium by co-culturing the first leukocyte withthe second leukocyte, the first leukocyte can be allogeneic orxenogeneic to the second leukocyte. In such embodiment, it is alsocontemplated that the first leukocyte be autologous, syngeneic,allogeneic or xenogeneic to a treated subject who is going to receivethe acellular preparation. In addition, it is also contemplated that thesecond leukocyte be autologous, syngeneic, allogeneic or xenogeneic to atreated subject who is going to receive the acellular preparation.

Once the conditioned medium or the conditioned blood has been obtainedit is further processed to substantially remove the cells and cellulardebris that can be present. This processing step can be achieved bysubmitting the conditioned medium or the conditioned blood to acentrifugation step and/or a filtration step. Since the majority of theimmuno-modulatory effects of the acellular preparations reside in afraction sensitive to ribonucleic acid degradation (e.g. RNasedegradation), this process step should be conducted in conditions whichwould substantially limit or even inhibit ribonucleic acid degradation.

The conditioned medium or the conditioned blood is also processed(preferably after the cell/cellular debris) so as to provide anenrichment in at least one miRNA species, and preferably a plurality ofmiRNA species. As used in the context of this invention, the term“enrichment” refers to the step of increasing the concentration of oneor more miRNA species in the acellular preparation when compared toconditioned medium/blood. In an embodiment, the term enrichment refersto the step of increasing, in the acellular preparation, theconcentration but not the relative abundance of the miRNA speciespresent in the conditioned medium/blood. In still another embodiment,the enrichment step can comprises substantially isolating the miRNAspecies from other components that may be present the conditionedmedium/blood (e.g. proteins such as cytokines for example). Thisenrichment step can be completed using various methods known to thoseskilled in the art, for example, chromatography, precipitation, etc.Since most of the immuno-modulatory effects of the acellularpreparations reside in a fraction sensitive to ribonucleic aciddegradation (e.g. RNase degradation), this process step should beconducted in conditions which would substantially limit or even inhibitribonucleic acid degradation.

The conditioned medium or the conditioned blood can also be processed tosubstantially remove the protein components (including the cytokines)and/or the deoxyribonucleic acid components that may be present. Suchfurther purification step can be made by using proteinase (to provide aprotein-free acellular preparation), DNAse (to provide a DNA-freeacellular preparation), chromatography or filtration (to provide afraction enriched in size-specific components present in the conditionedmedium/blood).

In some embodiments, it is also contemplated that the acellularpreparation be submitted to the selective enrichment in components ofthe conditioned medium/blood having a relative size equal to or lowerthan about 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4 kDa or 3 kDa.

Once the acellular preparation has been obtained, it can be formulatedfor administration to the subject. The formulation step can compriseadmixing the acellular preparation with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, and/or carriers.

The formulations are preferably in a liquid injectable form and caninclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength, additives such as albumin or gelatinto prevent absorption to surfaces. The formulations can comprisepharmaceutically acceptable solubilizing agents (e.g., glycerol,polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., thimerosal, benzyl alcohol,parabens), bulking substances or tonicity modifiers (e.g., lactose,mannitol).

In addition, if the acellular preparation is destined to be used toprevent an excessive immune reaction triggered by a vaccine, it can beformulated for administration with the vaccine. The acellularpreparation can be formulated for simultaneous administration with thevaccine by admixing the vaccine with the acellular preparation.Alternatively, the acellular preparation can be formulated foradministration prior to or after the vaccine, for example in aformulation that is physically distinct from the vaccine.

Further, if the acellular preparation is destined to be used to preventor limit an excessive immune reaction triggered by a transplant, it canbe formulated for administration prior to the transplantation. Theacellular preparations can be formulated for simultaneous administrationwith the transplant. Alternatively, the acellular preparations can beformulated for administration prior to or after the transplant. In anembodiment, the acellular preparation can be included in atransplantation medium or a preservation medium destined to receive thedonor cells or tissue. In such embodiment, the acellular preparation caninduce anergy and/or tolerance of the immune cells or stem cells presentin the cells/tissue intended to be transplanted.

Characterization of the miRNA Fraction of the Acellular Preparation

As shown herein, the miRNA fraction of the acellular preparation isassociated with the majority of the pro-tolerogenic immunomodulatoryeffects of the conditioned medium/blood. As also shown herein, thepro-tolerogenic immunomodulatory effects of the miRNA fraction of theacellular preparation are greatly reduced (and even abolished) when thecomponents of the conditioned blood/medium having an average molecularweight lower than about 10 kDa are removed or upon treatment with aribonucleic acid degradation agent (such as RNase A).

The acellular preparation described herein does comprise a plurality(also referred to a population) of distinct miRNA species whose relativeabundance differs from a control medium obtained from a control MLR(e.g. in which two allogeneic leukocyte populations are co-cultured) ora control blood obtained from administering unmodified allogeneicleukocytes to a test subject. The acellular preparation described hereinalso comprise a plurality (also referred to as a population) of distinctmiRNA species whose relative abundance differs from a conditioned mediumobtained from resting cells (e.g. a single cultured leukocytepopulation) or a blood obtained from a naïve test subject. Thismodulation in the relative abundance of the various miRNA species of theacellular preparation is believed to be tied to the pro-tolerogenicimmunomodulatory effects. The increased abundance of single miRNAspecies, unchanged abundance of single miRNA species and/or decreasedabundance of single miRNA species are believe to contribute to thepro-tolerogeneic immunomodulatory effects of the acellular preparation.In an embodiment, in the acellular preparation, the relative pattern ofexpression of the miRNA species present when compared to thecorresponding in the control conditioned medium/blood or medium fromresting cells/naïve blood is conserved.

In an embodiment, the acellular preparation comprises at least one miRNAspecies presented in FIG. 13. In another embodiment, the acellularpreparation comprises any combination of at least 2, 3, 4, 5, 10, 15,20, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 of the miRNAspecies presented in FIG. 13. In still another embodiment, the acellularpreparation comprises all the miRNA species presented in FIG. 13. FIG.13 provides the following miRNA species: hsa-let-7a-5p, hsa-let-7c,hsa-let-7d-5p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-miR-103a-3p,hsa-miR-105-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p,hsa-miR-128, hsa-miR-130a-3p, hsa-miR-132-3p, hsa-miR-134,hsa-miR-135a-5p, hsa-miR-135b-5p, hsa-miR-138-5p, hsa-miR-142-3p,hsa-miR-142-5p, hsa-miR-143-3p, hsa-miR-145-5p, hsa-miR-146a-5p,hsa-miR-147a, hsa-miR-148a-3p, hsa-miR-149-5p, hsa-miR-150-5p,hsa-miR-152, hsa-miR-155-5p, hsa-miR-15a-5p, hsa-miR-15b-5p,hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-181a-5p, hsa-miR-182-5p,hsa-miR-183-5p, hsa-miR-184, hsa-miR-185-5p, hsa-miR-186-5p,hsa-miR-187-3p, hsa-miR-18a-5p, hsa-miR-18b-5p, hsa-miR-191-5p,hsa-miR-194-5p, hsa-miR-195-5p, hsa-miR-196a-5p, hsa-miR-19a-3p,hsa-miR-19b-3p, hsa-miR-200a-3p, hsa-miR-203a, hsa-miR-205-5p,hsa-miR-206, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-21-5p, hsa-miR-210,hsa-miR-214-3p, hsa-miR-223-3p, hsa-miR-23b-3p, hsa-miR-26a-5p,hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-298,hsa-miR-299-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-302a-3p,hsa-miR-30b-5p, hsa-miR-30c-5p, hsa-miR-30e-5p, hsa-miR-31-5p,hsa-miR-325, hsa-miR-335-5p, hsa-miR-34a-5p, hsa-miR-363-3p,hsa-miR-379-5p, hsa-miR-383, hsa-miR-409-3p, hsa-miR-451a,hsa-miR-493-3p, hsa-miR-574-3p, hsa-miR-9-5p, hsa-miR-98-5p andhsa-miR-99b-5p.

In another embodiment, the acellular preparation comprises at least onemiRNA species whose relative abundance is increased when compared to acontrol medium/blood or resting cells/naïve blood. Such miRNA speciesare listed in Tables 1A to 1D.

TABLE 1A miRNA species whose relative abundance in the acellularpreparation is increased when compared to control medium/blood or mediumfrom resting cells/naïve blood as determined in FIG. 13. hsa-let-7a-5phsa-let-7c hsa-let-7e-5p hsa-miR-105-5p hsa-miR-130a-3p hsa-miR-132-3phsa-miR-134 hsa-miR-135a-5p hsa-miR-135b-5p hsa-miR-142-3phsa-miR-142-5p hsa-miR-147a hsa-miR-149-5p hsa-miR-155-5p hsa-miR-15a-5phsa-miR-181a-5p hsa-miR-187-3p hsa-miR-18a-5p hsa-miR-18b-5phsa-miR-200a-3p hsa-miR-205-5p hsa-miR-206 hsa-miR-21-5p hsa-miR-210hsa-miR-214-3p hsa-miR-27a-3p hsa-miR-27b-3p hsa-miR-298 hsa-miR-299-3phsa-miR-29b-3p hsa-miR-302a-3p hsa-miR-31-5p hsa-miR-34a-5p hsa-miR-383hsa-miR-451a hsa-miR-493-3p hsa-miR-574-3p hsa-miR-9-5p

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 1A. In still a further embodiment, theacellular preparation comprises a combination of at least 2, 3, 4, 5,10, 15, 20, 25, 30, 35 or 37 of any one of the miRNA species listed inTable 1A. In yet a further embodiment, the acellular preparationcomprises all the miRNA species listed in Table 1A.

TABLE 1B miRNA species whose relative abundance in the acellularpreparation is increased when compared to control medium/blood and whoserelative abundance in the control medium/blood is decreased whencompared the medium from resting cells/naïve blood as determined in FIG.13. miRNA species identified with an * show a log₂ fold regulationchange or a p ≤ 0.05 on a volcano plot. miRNA species identified with a# are identified on the volcano plots of FIG. 12. hsa-let-7a-5p*#hsa-let-7e-5p*# hsa-miR-132-3p* hsa-miR-21-5p* hsa-miR-27a-3p*hsa-miR-27b-3p* hsa-miR-298*# hsa-miR-34a-5p*#

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 1B. In still a further embodiment, theacellular preparation comprises a combination of at least 2, 3, 4, 5, 6or 7 of any one of the miRNA species listed in Table 1B. In yet afurther embodiment, the acellular preparation comprises all the miRNAspecies listed in Table 1B.

In an embodiment, the acellular preparation comprises at least one (orany combination of) miRNA species listed in Table 1B and showing a log₂fold regulation change or a p≤0.05 on a volcano plot (e.g.,hsa-let-7a-5p, hsa-let-7e-5p, hsa-miR-132-3p, hsa-miR-21-5p,hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-298 and/or hsa-miR-34a-5p). Instill another embodiment, the acellular preparation comprises at leastone (or any combination of) miRNA species listed in Table 1B andidentified on the volcano plots of FIG. 12 (e.g. hsa-let-7a-5p,hsa-let-7e-5p, hsa-miR-298 and/or hsa-miR-34a-5p).

TABLE 1C miRNA species whose relative abundance in the acellularpreparation is increased when compared to the medium/blood from restingcells/naïve blood and whose relative abundance in the controlblood/medium is increased when compared to the medium from restingcells/naïve blood as determined in FIG. 13. miRNA species identifiedwith an * show a log₂ fold regulation change or a p ≤ 0.05 on a volcanoplot. miRNA species identified with a # are identified on the volcanoplots of FIG. 12. hsa-let-7c hsa-miR-105-5p hsa-miR-130a-3p hsa-miR-134#hsa-miR-135a-5p hsa-miR-135b-5p* hsa-miR-142-3p hsa-miR-142-5phsa-miR-147a* hsa-miR-149-5p* hsa-miR-155-5p* hsa-miR-15a-5phsa-miR-181a-5p hsa-miR-187-3p hsa-miR-18a-5p hsa-miR-18b-5phsa-miR-200a-3p hsa-miR-205-5p hsa-miR-206* hsa-miR-210 hsa-miR-214-3p*hsa-miR-299-3p hsa-miR-29b-3p hsa-miR-302a-3p* hsa-miR-31-5p hsa-miR-383hsa-miR-451a hsa-miR-493-3p hsa-miR-574-3p# hsa-miR-9-5p*

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 1C. In still a further embodiment, theacellular preparation comprises a combination of at least 2, 3, 4, 5,10, 15, 20, 25, or 29 of any one of miRNA species listed in Table 1C. Inyet a further embodiment, the acellular preparation comprises all themiRNA species listed in Table 1C.

In an embodiment, the acellular preparation comprises at least one (orany combination of) miRNA species listed in Table 1C and showing a log₂fold regulation change or a p≤0.05 on a volcano plot (e.g.hsa-miR-135b-5p, hsa-miR-147a, hsa-miR-149-5p, hsa-miR-155-5p,hsa-miR-206, hsa-miR-214-3p, hsa-miR-302a-3p and/or hsa-miR-9-5p). Instill another embodiment, the acellular preparation comprises at leastone (or any combination of) miRNA species listed in Table 1C andidentified on the volcano plots of FIG. 12 (e.g. hsa-miR-134 and/orhsa-miR-574-3p).

TABLE 1D Selection of the miRNA species from Table 1E which show a log₂fold regulation change or a p ≤ 0.05 on a volcano plot. hsa-miR-135b-5phsa-miR-147a hsa-miR-149-5p hsa-miR-155-5p hsa-miR-206 hsa-miR-214-3phsa-miR-302a-3p hsa-miR-9-5p

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 1D. In still a further embodiment, theacellular preparation comprises a combination of at least 2, 3, 4, 5, 6or 7 of any one of miRNA species listed in Table 1D. In yet a furtherembodiment, the acellular preparation comprises all the miRNA specieslisted in Table 1D.

In another embodiment, the acellular preparation comprises at least onemiRNA species whose relative abundance is decreased when compared to acontrol medium/blood or the medium from resting cells/naïve blood. SuchmiRNA species are listed in Tables 2A to 2D.

TABLE 2A miRNA species whose relative abundance in the acellularpreparation is decreased when compared to control medium/blood or mediumfrom resting cells/naïve blood as determined in FIG. 13. hsa-let-7d-5phsa-let-7g-5p hsa-miR-103a-3p hsa-miR-125a-5p hsa-miR-125b-5phsa-miR-126-3p hsa-miR-128 hsa-miR-138-5p hsa-miR-143-3p hsa-miR-145-5phsa-miR-146a-5p hsa-miR-148a-3p hsa-miR-150-5p hsa-miR-152hsa-miR-15b-5p hsa-miR-16-5p hsa-miR-17-5p hsa-miR-182-5p hsa-miR-183-5phsa-miR-184 hsa-miR-185-5p hsa-miR-186-5p hsa-miR-191-5p hsa-miR-194-5phsa-miR-195-5p hsa-miR-196a-5p hsa-miR-19a-3p hsa-miR-19b-3phsa-miR-203a hsa-miR-20a-5p hsa-miR-20b-5p hsa-miR-223-3p hsa-miR-23b-3phsa-miR-26a-5p hsa-miR-26b-5p hsa-miR-29c-3p hsa-miR-30b-5phsa-miR-30c-5p hsa-miR-30e-5p hsa-miR-325 hsa-miR-335-5p hsa-miR-363-3phsa-miR-379-5p hsa-miR-409-3p hsa-miR-98-5p hsa-miR-99b-5p

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 2A. In still a further embodiment, theacellular preparation comprises a combination of at least 2, 3, 4, 5,10, 15, 20, 25, 30, 35, 40, 45 or 46 of any one of miRNA species listedin Table 2A. In yet a further embodiment, the acellular preparationcomprises all the miRNA species listed in Table 2A.

TABLE 2B miRNA species whose relative abundance in the acellularpreparation is decreased when compared to control medium/blood and whoserelative abundance in the control medium/blood is increased whencompared the medium from resting cells/naïve blood as determined in FIG.13. miRNA species identified with an * show a log₂ fold regulationchange or a p ≤ 0.05 on a volcano plot. hsa-miR-183-5p* hsa-miR-203a*hsa-miR-325 hsa-miR-363-3p*

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 2B. In still a further embodiment, theacellular preparation comprises a combination of at least 2 or 3 of anyone of the miRNA species listed in Table 2B. In yet a furtherembodiment, the acellular preparation comprises all the miRNA specieslisted in Table 2B.

In an embodiment, the acellular preparation comprises at least one miRNAspecies (or any combination thereof) listed in Table 2B and showing alog₂ fold regulation change or a p≤0.05 on a volcano plot (e.g.hsa-miR-183-5p, hsa-miR-203a and/or hsa-miR-363-3p).

TABLE 2C Selection of the miRNA species from Table 2B which show a log₂fold regulation change or a p ≤ 0.05 on a volcano plot. hsa-miR-183-5phsa-miR-203a hsa-miR-363-3p

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 2C. In still a further embodiment, theacellular preparation comprises a combination of at least 2 of any oneof miRNA species listed in Table 2C. In yet a further embodiment, theacellular preparation comprises all the miRNA species listed in Table2C.

TABLE 2D miRNA species whose relative abundance in the acellularpreparation is decreased when compared to the medium from restingcells/naïve blood and whose relative abundance in the controlblood/medium is decreased when compared to the medium from restingcells/naïve blood in FIG. 13. miRNA species identified with an * show alog₂ fold regulation change or a p ≤ 0.05 on a volcano plot. miRNAspecies identified with a # are identified on the volcano plots of FIG.12. hsa-let-7d-5p hsa-let-7g-5p hsa-miR-103a-3p hsa-miR-125a-5phsa-miR-125b-5p# hsa-miR-126-3p hsa-miR-128 hsa-miR-138-5phsa-miR-143-3p hsa-miR-145-5p hsa-miR-146a-5p hsa-miR-148a-3p#hsa-miR-150-5p hsa-miR-152 hsa-miR-15b-5p hsa-miR-16-5p hsa-miR-17-5phsa-miR-182-5p hsa-miR-184 hsa-miR-185-5p hsa-miR-186-5p hsa-miR-191-5phsa-miR-194-5p hsa-miR-195-5p hsa-miR-196a-5p# hsa-miR-19a-3phsa-miR-19b-3p hsa-miR-20a-5p hsa-miR-20b-5p hsa-miR-223-3phsa-miR-23b-3p hsa-miR-26a-5p hsa-miR-26b-5p hsa-miR-29c-3phsa-miR-30b-5p hsa-miR-30c-5p hsa-miR-30e-5p hsa-miR-335-5phsa-miR-379-5p hsa-miR-409-3p hsa-miR-98-5p hsa-miR-99b-5p

In a further embodiment, the acellular preparation comprises at leastone miRNA species listed Table 2D. In still a further embodiment, theacellular preparation comprises a combination of at least 2, 3, 4, 5,10, 15, 20, 25, 30, 35, 40 or 41 of any one of miRNA species listed inTable 2D. In yet a further embodiment, the acellular preparationcomprises all the miRNA species listed in Table 2D.

In an embodiment, the acellular preparation comprises at least one miRNAspecies (or any combination thereof) listed in Table 2D and areidentified on the volcano plots of FIG. 12 (e.g. hsa-miR-125b-5p,hsa-miR-148a-3p and/or hsa-miR-196a-5p).

It is contemplated that the acellular preparation comprises at least one(and in an embodiment any combination of) miRNAs species from any one ofTables 1A to 1D and at least one (and in an embodiment any combinationof) miRNAs species from any one of Tables 2A to 2D.

In yet another embodiment, the acellular preparation can comprise atleast one of the miRNA species identified in the volcano plots of FIG.12. For example, the acellular preparation can comprise at least one (orany combination of) miRNA species from the following list: has-miR-298,has-miR-34a-5p, has-miR-574-3p, has-miR-125b-5p, has-let-7a-5p,has-miR-196a-5p, has-miR-148a-3p, has-let-7e-5p and has-miR-134. Instill another embodiment, the acellular preparation can comprise atleast one (or any combination of) miRNA species identified on FIG. 12and having a relative abundance which is increased in the acellularpreparation when compared to the control medium/blood (e.g. miR-298,has-miR-34a-5p, has-miR-574-3p, has-let-7a-5p, has-miR-196a-5p,has-miR-148a-3p, has-let-7e-5p and/or has-miR-134). In still anotherembodiment, the acellular preparation can comprise the miRNA speciesidentified on FIG. 12 and having a relative abundance which is increasedin the acellular preparation and the control medium/blood when comparedto the resting cells/naïve blood (e.g. has-miR-125b-5p).

In yet another embodiment, the acellular preparation comprises at leastone (or any combination of) miRNA species presented on FIG. 12A whichexhibits at least a log₂ fold modulation in abundance (e.g. miR-302a-3p,miR214-3p, miR-147a, miR206, miR 155-5p and/or miR-9-5p). In yet stillanother embodiment, the acellular preparation comprises at least one (orany combination of) of miRNA species presented on FIG. 12A whichexhibits at least p≤0.05 (e.g. miR214-3p, miR-147a, miR206, miR 155-5pand/or miR-9-5p). In yet another embodiment, the acellular preparationcomprises at least one (or any combination of) miRNA species presentedon FIG. 12B which exhibits at least a log₂ fold modulation in abundance(e.g. miR-149-5p and/or miR-214-3p). In yet still another embodiment,the acellular preparation comprises the miRNA species presented on FIG.12B which exhibits at least p≤0.05 (e.g. miR-214-3p). In yet anotherembodiment, the acellular preparation comprises at least one (or anycombination of) miRNA species presented on FIG. 12C which exhibits atleast a log₂ fold modulation in abundance (e.g. miR-147a, miR-183-5p,miR-9-5p and/or miR-155-5p). In yet still another embodiment, theacellular preparation comprises at least one (or any combination of)miRNA species presented on FIG. 12C which exhibits at least p≤0.05 (e.g.miR-9-5p and/or miR-155-5p).

It is contemplated that the acellular preparation comprises at least one(and in an embodiment any combination of) miRNAs species from any one ofTables 1A to 1D, at least one (and in an embodiment any combination of)miRNAs species from any one of Tables 2A to 2D and at least one (and inan embodiment any combination of) miRNA species identified in any one ofthe FIG. 12A to 12C.

Methods for Modulating the Treg/Pro-Inflammatory T Cells Ratio

The present invention also provides methods and acellular preparationsfor increasing the ratio of the level of regulatory T cells with respectto the level of pro-inflammatory T cells. In the present invention, theratio can be increased either by augmenting the level of regulatory Tcells in the subject or decreasing the level of pro-inflammatory T cellsin the treated subject. Alternatively, the ratio can be increased byaugmenting the level of regulatory T cells in the subject and decreasingthe level of pro-inflammatory T cells in the treated subject. When theTreg/pro-inflammatory T cells ratio is increased in the treated subject,it is considered that a state of anergy and/or of increased tolerance isinduced or present in the treated subject. The induction of a state ofanergy or immunotolerance in individuals experiencing an abnormallyelevated immune reaction can be therapeutically beneficial for limitingthe symptoms or pathology associated with the abnormally elevated immunereaction. In some embodiments, it is not necessary to induce a state ofcomplete anergy or tolerance, a partial induction of anergy or tolerancecan be beneficial to prevent, treat and/or alleviate the symptoms of adisorder associated with a pro-inflammatory state (such as, for example,an auto-immune disease or an excessive immune response).

In order to increase the Treg/pro-inflammatory T cells ratio, theacellular preparation is administered to the treated subject in atherapeutically effective amount. In a first embodiment, the acellularpreparation can be prepared using the conditioned blood obtained byadministering a first leukocyte to a test subject. In such embodiment,the first leukocyte, allogeneic or xenogeneic to the test subject, canbe allogeneic, xenogeneic, autologous or syngeneic to the treatedsubject. In a second embodiment, the acellular preparation can beprepared using the conditioned medium obtained by co-culturing a firstleukocyte with a second leukocyte. In such embodiment, the firstleukocyte, allogeneic or xenogeneic to the second leukocyte, can beallogeneic, xenogeneic, autologous or syngeneic to the treated subject.In addition, the second leukocyte, allogeneic or xenogeneic to the firstleukocyte, can be allogeneic, xenogeneic, autologous or syngeneic to thetreated subject.

As shown herein, the administration of the acellular preparation inducesa state of anergy or immune tolerance in the treated subject. In someembodiments, the state of anergy can persist long after theadministration of the acellular preparation (as shown below, at least270 days in mice). In an optional embodiment, the state of anergy doesnot revert back to a pro-inflammatory state upon a challenge with, forexample, an immunogen (such as an immunogenic or allogeneic cell).Consequently, the methods and cellular preparations described herein areuseful for the treatment, prevention and/or alleviation of symptomsassociated with abnormal/excessive immune responses and conditionsassociated thereto.

Autoimmunity arises consequent to an animal/individual's immune systemrecognizing their own tissues as “non-self”. Autoimmunity is largely acell-mediated disease with T lymphocytes playing a central role in“self” recognition and are, in many cases, also the effector cells. TheNon-Obese Diabetic (NOD) mouse is an inbred strain that exhibits thespontaneous development of a variety of autoimmune diseases includinginsulin dependent diabetes. It is considered to be an exemplary mousemodel of autoimmunity in general. The murine autoimmune diabetesdevelops beginning around 10 to 15 weeks of age and has been extensivelyused to study the mechanisms underlying autoimmune-mediated diabetes,therapeutic interventions and the effect of viral enhancers on diseasepathogenesis. Diabetes develops in NOD mice as a result of insulitis, aleukocytic infiltrate of the pancreatic islets. This can be exacerbatedif mice are exposed to killed mycobacterium or other agents (Coxsackievirus for example). Multiple studies have established that thepathogenesis of diabetes in the NOD mouse is very similar to thatobserved in human type I diabetes (T1D) in that it is characterized bythe breakdown of multiple tolerance pathways and development of severeinsulitis of the islets prior to β-cell destruction. Moreover, T cells(including Th1, Th17 and Tregs) have been identified as key mediators ofthe autoimmune disease process though other cells (NK cells, B-cells, DCand macrophages) are also observed. Indeed, the NOD mouse model hastranslated into successful clinical human trials utilizing T-celltargeting therapies for treatment of many autoimmune diseases, includingT1D. The loss of function arising from pro-inflammatory allo-recognitionis exemplified by the destruction of the islets of Langerhans (insulinsecreting β cells) in the pancreas of the NOD mice leading to the onsetof Type 1 diabetes. In the context of type I diabetes, pro-tolerogenicallo-recognition is going to confer the protection and survival of theislets of Langerhans and the inhibition of diabetes in the treatedsubject.

Current treatment of most autoimmune diseases is problematic since itfocuses on addressing disease symptoms, not causation. Typically,treatment for chronic autoimmune disease is via systemic steroid (e.g.,dexamethasone) administration to induce a general immunosuppression andto act as an anti-inflammatory agent. It is believed that one mechanismof this immunosuppression may be the induction of Treg cells. Inaddition to steroids, the administration of IVIg (pooled, polyvalent,IgG purified from the plasma of >1 000 blood donors) can alsoeffectively treat some autoimmune diseases including immunethrombocytopenia (ITP). Interestingly, the onset of diabetes in NOD micecan also be delayed, but not fully blocked by administration of IVIg andthis may correlate with induction of Tregs (and/or IL-10). Moreoveretanercept (trade name ENBREL®), a soluble TNF-receptor, has also beenshown to decrease the incidence of diabetes in NOD mice and has beenused in small scale human trials. Hence, novel approaches to increaseTreg cells (and/or IL-10) while decreasing inflammatory T cell responses(e.g., Th17, NK cells) could be beneficial in treating autoimmunediabetes.

A state of anergy or immune tolerance can be considered therapeuticallybeneficial in subjects experiencing (or at risk of experiencing) anabnormal immune response, such as for example an auto-immune disease.Individuals afflicted by auto-immune diseases have either low levels ofTregs and/or elevated levels of pro-inflammatory T cells (such as Th17and/or Th1) when compared to age- and sex-matched healthy individuals.Such auto-immune diseases include, but are not limited to, type Idiabetes, rheumatoid arthritis, multiple sclerosis, lupus, immunethrombocytopenia, experimental autoimmune encephalomyelitis, auto-immuneuveitis, psoriasis inflammatory bowel disease, scleroderma and Crohn'sdisease. Because it is shown herein that the acellular preparations arebeneficial for increasing the ratio Tregs/pro-inflammatory T cells, itis expected that administration of the acellular preparations toafflicted subjects will alleviate symptoms associated with theauto-immune disease and/or prevent disease severity.

A state of anergy or tolerance can also be considered therapeuticallybeneficial in subjects at risk of developing an abnormallyelevated/excessive immune response. Such abnormally elevated immuneresponse can be observed in subjects receiving a vaccine. For example,it has been shown that subjects receiving a respiratory syncytial virus(RSV) vaccine develop an excessive immune response. Because it is shownherein that the acellular preparations are beneficial for increasing theratio Tregs/pro-inflammatory T cells, it is expected that administrationof the acellular preparations to subject having received or intended toreceive a vaccine will alleviate symptoms associated with theadministration of the vaccine and/or prevent the development of anexcessive immune response. In such embodiment, the acellular preparationcan be administered (or formulated for administration) prior to thevaccine, simultaneously with the vaccine or after the administration ofthe vaccine. When used to prevent or limit excessive immune response toa vaccine, the acellular preparations can be manufactured from aconditioned medium. The conditioned medium can be obtained byco-culturing a first leukocyte, being allogeneic or xenogeneic to asecond leukocyte, which can be allogeneic, xenogeneic, autologous orsyngeneic to the subject to be vaccinated. The second leukocyte, muchlike the first leukocyte, can be allogeneic, xenogeneic, autologous orsyngeneic to the subject to be vaccinated. When used to prevent or limitan excessive immune response to a vaccine, the acellular preparationscan also be manufactured from a conditioned blood. The conditioned bloodcan be obtained by administered a first leukocyte, being allogeneic orxenogeneic to the test subject, which can be allogeneic, xenogeneic,autologous or syngeneic to the subject to be vaccinated.

Such abnormally elevated immune response can also be observed insubjects having received a transplant (cells or tissues). In theseinstances, the acellular preparations can be used to prevent or limitthe elevated/excessive immune response (e.g. graft destruction or graftrejection). In an embodiment, the acellular preparation can be contactedwith the cells/tissue to be transplanted prior to the transplantation(e.g. for example in a transplant medium or a preservation medium). Whenused to prevent or limit graft destruction or graft rejection, theacellular preparations can be manufactured from a conditioned medium.The conditioned medium can be obtained by co-culturing a firstleukocyte, being allogeneic or xenogeneic to a second leukocyte, whichcan be allogeneic, xenogeneic, autologous or syngeneic to the subject tobe treated. Alternatively, the first leukocyte is allogeneic,xenogeneic, autologous or syngeneic to the cells or tissue intended tobe grafted. The second leukocyte, much like the first leukocyte, can beallogeneic, xenogeneic, autologous or syngeneic to the subject to betreated. Alternatively, the second leukocyte is allogeneic, xenogeneic,autologous or syngeneic to the cells or tissue intended to be grafted.When used to prevent or limit graft destruction or graft rejection, theacellular preparations can also be manufactured from a conditionedblood. The conditioned blood can be obtained by administering a firstleukocyte, being allogeneic or xenogeneic to the test subject, which canbe allogeneic, xenogeneic, autologous or syngeneic to the subject to betreated. Alternatively, the first leukocyte is allogeneic, xenogeneic,autologous or syngeneic to the cells or tissue intended to be grafted.

Alternatively or optionally, the acellular preparations can also be usedto prevent or limit a graft-vs.-host disease (GVHD) in a subject havingreceived or intended to receive transplanted immune cells or stem cells.In an embodiment, the acellular preparations can be contacted (e.g.cultured) with the cells intended to be grafted prior to transfusion inthe subject (e.g. for example in a transplantation medium orpreservation medium) to induce a state of anergy or tolerance in thosecells. In another embodiment, the acellular preparations can beadministered to the subject prior to the transfusion of immune/stemcells to induce a state of anergy or tolerance to prevent or limit GVHD.In still another embodiment, the acellular preparations can beadministered simultaneously with the transfused immune/stem cells toprevent or limit GVHD. In yet another embodiment, the acellularpreparations can be administered to a subject having been transfusedwith immune cells or stem cells either to alleviate the symptomsassociated to GVHD (when the subject experiences such symptoms) or toprevent GVHD (when the subject is at risk of experiencing suchsymptoms).

For the treatment of GVHD, the conditioned medium can be obtained byco-culturing two allogeneic/xenogeneic leukocyte population. In anembodiment, the first leukocyte population can be allogeneic,xenogeneic, syngeneic to or derived from the donor (of the immune orstem cells). In another embodiment, the first leukocyte population canbe allogeneic, xenogeneic, syngeneic to or derived from the recipient(intended to receive the immune or stem cells). In still anotherembodiment, the second leukocyte population can be allogeneic,xenogeneic, syngeneic to or derived from the donor. In yet anotherembodiment, the second leukocyte population can be allogeneic,xenogeneic, syngeneic to or derived from the recipient. For thetreatment of GVHD, the conditioned blood can be obtained byadministering a first leukocyte allogeneic or xenogeneic to the testsubject (e.g. and in an embodiment to the donor). In an embodiment, thefirst leukocyte population can be allogeneic, xenogeneic, syngeneic toor derived from the donor. In another embodiment, the first leukocytepopulation can be allogeneic, xenogeneic, syngeneic to or derived fromthe recipient.

The acellular preparation the acellular preparation can be administered(or formulated for administration) prior to the transplant,simultaneously with the transplant or after the transplant.

In the methods and acellular preparations described herein, it iscontemplated that the acellular-based preparations be optionallyadministered with other therapeutic agents known to be useful for thetreatment, prevention and/or alleviation of symptoms of conditionsassociated to an excessive/abnormal immune response, such as, forexample, cortisone, IL-10, IL-11 and/or IL-12.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—Material and Methods

Human PBMC and Dendritic Cell Preparation.

Human whole blood was collected in heparinized vacutainer bloodcollection tubes (BD, Franklin Lakes, N.J.) from healthy volunteerdonors following informed consent. PBMC were isolated from diluted wholeblood using FicollePaque PREMIUM™ (GE Healthcare Bio-Sciences Corp,Piscataway, N.J.) as per the product instructions. The PBMC layer waswashed twice with 1× Hank's Balanced Salt Solution (HBSS; without CaCl₂and MgSO₄; Invitrogen by Life Technologies, Carlsbad, Calif.) andresuspended in the appropriate media as needed for mixed lymphocytereactions and flow cytometric analysis of Treg and Th17 phenotypes.Dendritic cells (DC) were prepared from isolated PBMC as described byO'Neill and Bhardwaj (O'Neill et al., 2005). Briefly, freshly isolatedPBMC were overlaid on Petri dishes for 3 h in AIM V serum free culturemedium (Invitrogen, Carlsbad, Calif.). Non-adherent cells were gentlywashed off the plate. The adherent cells (monocyte rich cells) weretreated with IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&DSystems, Minneapolis, Minn.) in AIM V medium. Cells were again treatedwith IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifugedand resuspended in fresh media supplemented with DC maturation factors(TNF-α, IL-1β, IL-6; R&D Systems, Minneapolis, Minn.) and prostaglandinE2 (Sigma Aldrich, St. Louis, Mo.). The mature DC-like cells wereharvested on day 7 and CD80, CD83, CD86 and HLA-DR expressions weredetermined to confirm DC maturation via flow cytometry (FACSCalibur™Flow Cytometer, BD Biosciences, San Jose, Calif.).

Murine Splenocyte and Tissue Harvesting.

All murine studies were done in accordance with the Canadian Council ofAnimal Care and the University of British Columbia Animal Care Committeeguidelines and were conducted within the Centre for Disease Modeling atthe University of British Columbia. Murine donor cells used for the invivo donation and in vitro studies were euthanized by CO₂. Threeallogeneic strains of mice were utilized for syngeneic and allogeneic invitro and in vivo challenge: Balb/c, H-2^(d); C57Bl/6, H-2^(b); and C3H,H-2^(k). Murine spleens, brachial lymph nodes, and peripheral blood werecollected at the indicated days. Mouse spleens and brachial lymph nodeswere dissected and placed into cold phosphate buffered saline (PBS; 1.9mM NaH₂PO₄, 8.1 mM Na₂HPO₄, and 154 mM NaCl, pH 7.3) containing 0.2%bovine serum albumin (BSA; Sigma Aldrich, St. Louis, Mo.) and kept onice until ready to process. Whole blood was collected in heparinizedtubes via cardiac puncture. Murine donor splenocytes were prepared fromfreshly harvested syngeneic or allogeneic spleens via homogenizationinto a cell suspension in PBS (0.2% BSA) using the frosted end of twomicroscope slides. The resultant cell suspension was spun down at 500×g.The splenocyte pellet was resuspended in 1 mL of 1×BD Pharm LYSE™ lysingbuffer (BD Biosciences, San Diego, Calif.) and incubated for 1 min atroom temperature. Lymph node cells were harvested via tissuehomogenization as described above, washed twice and resuspended in PBS(0.2% BSA) for flow cytometric analysis of Th17, Treg and murinehaplotype. Recipient peripheral blood lymphocytes were prepared vialysis of the red cells (BD Pharm Lyse lysing buffer; BD Biosciences, SanDiego, Calif.) at 1× concentration, followed by washing (1×) andresuspension in PBS (0.2% BSA) for flow analysis of Th17, Treg andmurine haplotype.

mPEG Modification (PEGylation) of PBMCs and Splenocytes.

Human PBMC and murine splenocytes were derivatized usingmethoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA; LaysanBio Inc. Arab, Ala.) with a molecular weight of 20 kDa as previouslydescribed (Scott et al., 1997; Murad et al, 1999A; Chen et al., 2003;Chen et al., 2006, Wang et al., 2011). Grafting concentrations was 1 mMper 4×10⁶ cells/mL. Cells were incubated with the activated mPEG for 60min at room temperature in isotonic alkaline phosphate buffer (50 mMK₂HPO₄ and 105 mM NaCl; pH 8.0), then washed twice with 25 mM HEPES/RPMI1640 containing 0.01% human albumin. Murine splenocytes used for in vivostudies were resuspended in sterile saline at a final cell density of2.0×10⁸ cells/ml for intravenous (i.v.) injection.

In Vitro and In Vivo Cell Proliferation.

Cell proliferation (both in vitro and in vivo) was assessed via flowcytometry using the CELLTRACE™ CFSE (Carboxyfluorescein diacetate,succinimidyl ester) Cell Proliferation Kit (Invitrogen by LifeTechnologies e Molecular probes, Carlsbad, Calif.). Human and murinecells labeling was done according to the product insert at a finalconcentration of 2.5 mM CFSE per 2×10⁶ cells total. Donor and recipientcell proliferation was differentially determined by haplotype analysis.In some experiments, cell proliferation was measured by ³H-thymidineincorporation. In these experiments, donor splenocytes (5.12×10⁶ cellsper well) were co-incubated in triplicate in 96-well plates at 37° C.,5% CO₂ for 3 days. On day 3, all wells were pulsed with ³H-thymidine andincubated for 24 h at 37° C., 5% CO₂. Cellular DNA was collected onfilter mats using a Skatron cell harvester (Suffolk, U.K.) and cellularproliferation was measured by ³H-thymidine incorporation.

Mixed Lymphocyte Reaction (MLR)—Control and Conditioned Medium.

The immunodulatory effects of the various preparations were assayedusing a MLR (Murad et al, 1999A; Chen et al., 2003; Chen et al., 2006;Wang et al., 2011). For the human MLRs, PBMC from two MHC-disparatehuman donors were labeled with CFSE. For mice MLR, splenocytes from twoH-2-disparate mice (Balb/c and C57Bl/6) were labeled with CFSE. Each MLRreaction well contained a total of 1×10⁶ cells (single donor for restingor mitogen stimulation or equal numbers for disparate donors for MLR).Cells were plated in multiwell flat-bottom 24-well tissue culture plates(BD Biosciences, Discovery Labware, Bedford, Mass.). PBMC proliferation,cytokine secretion, as well as Treg and Th17 phenotyping was done. Forflow cytometric analysis, the harvested cells were resuspended in PBS(0.1% BSA).

Immunophenotyping by Flow Cytometry.

The T lymphocytes populations (double positive for CD3⁺ and CD4⁺) weremeasured by flow cytometry using fluorescently labeled anti-CD3 andanti-CD4 (BD Pharmingen, San Diego, Calif.), anti-IL-2, anti-IL-4,anti-IL-10, anti-IL-12, anti-IL-17, anti-FoxP3, anti-NK1.1, anti-IFN-γ,anti-TNF-α, anti-CD152, anti CD62L and anti-CD11c monoclonal antibodies.Human and mouse Regulatory T lymphocytes (Treg) were CD3⁺/CD4⁺ andFoxP3⁺ (transcription factor) while inflammatory Th17 lymphocytes cellswere CD3⁺/CD4⁺ and IL-17⁺ (cytokine) as measured per the BD Treg/Th17Phenotyping Kit (BD Pharmingen, San Diego, Calif.). After the staining,the cells (1×10⁶ cells total) were washed and resuspended in PBS (0.1%BSA) prior to flow acquisition. Isotype controls were also used todetermine background fluorescence. All samples were acquired using theFACSCalibur™ flow cytometer (BD Biosciences, San Jose, Calif.) andCellQuest Pro™ software for both acquisition and analysis.

In Vivo Murine Studies.

The following strains were used Balb/c, H-2^(d); C57Bl/6, H-2^(b); andC3H, H-2^(k) (Chen et al., 2003; Chen et al., 2006) as well a NOD(Anderson et al., 2005). All mice (donors and recipients) were 9-11weeks old. Donor splenocytes were prepared were transfused intravenously(i.v.) via the tail vein into recipient animals. BALB/c and C57BL/6 miceinjected with sterile saline served as control animals. Animals wereeuthanized by CO₂ at predetermined intervals at which time blood,brachial lymph nodes and spleen were collected and processed forTh17/Treg phenotyping analysis and splenocyte proliferation studies byflow cytometry.

Conditioned Plasma.

Mouse were either untreated (naïve) or treated with saline, non-polymermodified allogeneic splenocytes or PEGylated allogeneic splenocytes(obtained by the procedures explained above). After five days, acell-free conditioned plasma was obtained (from mouse blood using themirVana™ PARIS™ kit from Ambion by Life Technologies) and transfused toanother naïve mouse.

Plasma Fractionation.

The plasma fractionation was performed using centrifugal filtermolecular cutoff devices. Millipore's Amicon® Ultra-0.5 centrifugalfilter devices were used (Amicon Ultra 3k, 10K, 30K, 50K, and 100Kdevices).

miRNA Extraction.

The miRNA was extracted from samples (conditioned medium or plasma)using mirVana™ PARIS™ kit from Ambion® by Life Technologies according tothe manufacturer's instructions. Briefly, the sample is mixed with the2× denaturing solution provided and subjected to acid-phenol:chloroformextraction. To isolate RNA that is highly enriched for small RNAspecies, 100% ethanol was added to bring the samples to 25% ethanol.When this lysate/ethanol mixture was passed through a glass-fiberfilter, large RNAs are immobilized, and the small RNA species arecollected in the filtrate. The ethanol concentration of the filtrate wasthen increased to 55%, and it was passed through a second glass-fiberfilter where the small RNAs become immobilized. This RNA is washed a fewtimes, and eluted in a low ionic strength solution. Using this approach,an RNA fraction highly enriched in RNA species <200 nt can be obtained.Note that the large RNA species (>200 nt) can be recovered from thefirst filter if necessary.

TA Preparations.

The murine miRNA preparations (e.g. TA1 preparations) used wereextracted from the conditioned plasma obtained 5 days after mice havereceived mPEG allogeneic splenocytes. Extraction can occur at timepoints other than 5 days (e.g., 24 hours post administration) and yieldsimilar results (data not shown). Five days was chosen as Treg levelsachieved maximal levels at this point in the mice. The human miRNApreparations (e.g. TA2 preparations) used were extracted from theconditioned medium of an mPEG-MLR harvested 72 hours following theinitiation of the mPEG-MLR. However, miRNA harvested from human PBMCmPEG-MLR at 24 hours also yields the desired immunomodulatory effects(data not shown). To calibrate, miRNA concentration can be quantitatedvia a Qubit® 2.0 Fluorometer (LifeTechnologies) and selected fluorescentdyes which emit a signal only when bound to specific target (i.e.,miRNA) molecules.

miRNA Characterization.

The miRNA of the conditioned medium were characterized by qPCR using themiScript miRNA™ PCR Array Human Immunopathology (Qiagen) for humanconditioned medium and the Mouse Immunopathology miRNA PCR Array™(Qiagen) for mouse conditioned plasma/media.

RNase Treatment.

Murine plasma was pooled and for each individual mouse. For each 500 μLof murine plasma (or the <10 kDa plasma fraction), 50 ng RNase (RNase A,20 mg/mL stock, Life Technologies (In Vitrogen)) was added. Then sampleswere incubated for 10 minutes at 37° C. to degrade the nucleic acids.The control plasma (or <10 kDa fraction) without RNAase A treatment wasincubated at 37° C. for 10 min. The RNase treated plasma (100 μl permouse) was injected (i.v.) into mice (n=5). RNase A alone (10 ng/mouse)was used for the control mice to insure that the RNase A was not toxicand this trace amount of RNase did not have an in vivo immunomodulatoryeffects.

Phosphorylation of Phosphokinases.

Analyzing the phosphorylation state of kinases and their proteinsubstrates allows for the characterization of the effects of conditionedplasma or media on how cells respond to allogeneic stimuli. The humanphospho-kinase array (R&D Systems Inc) is a rapid, sensitive tool tosimultaneously detect the relative levels of phosphorylation of 43kinase phosphorylation sites and 2 related total proteins. Each captureantibody was carefully selected using cell lysates prepared from celllines known to express the target protein. Capture and controlantibodies are spotted in duplicate on nitrocellulose membranes. Celllysates are diluted and incubated overnight with the humanphospho-kinase array. The array is washed to remove unbound proteinsfollowed by incubation with a cocktail of biotinylated detectionantibodies. Streptavidin-HRP and chemiluminescent detection reagents areapplied and a signal is produced at each capture spot corresponding tothe amount of phosphorylated protein bound.

Statistical Analysis.

Data analysis for flow analysis was conducted using SPSS™ (v12)statistical software (Statistical Products and Services Solutions,Chicago, Ill., USA). For significance, a minimum p value of <0.05 wasused. For comparison of three or more means, a one-way analysis ofvariance (ANOVA) was performed. When significant differences were found,a post-hoc Tukey test was used for pair-wise comparison of means. Whenonly two means were compared, student-t tests were performed.

Example II—In Vitro and In Vivo Immunomodulation of Size-FractionatedAcellular Preparations

Two-way human PBMC MLRs were prepared using the conditioned mediumcollected at 72 hours from mPEG MLR as the primary MLR. The conditionedmedium was fractionated with respect to its molecular weight (higher orlower than 10 kDa). As shown on FIG. 1, the fraction of the conditionedmedium derived from PEGylated MLR and having a molecular weight of lessthan 10 kDa retained the ability to increase human Treg levels in vitro.As also shown in FIG. 1, the fraction having a molecular weight higherthan 10 kDa did not have the ability to increase Treg levels in vitro inthe secondary MLR.

A conditioned cell-free plasma from untreated mouse (naïve), mousehaving received saline (saline), allogeneic unmodified splenocytes(allogeneic) and PEGylated allogeneic splenocytes (mPEG-allogeneic) wereobtained 5 days after treatment. The conditioned plasma was either leftuntreated (e.g. complete) or fractionated in function of the size of itscomponents (>100 kDa, between 30 and 100 kDa, between 10 and 30 kDa or<10 kDa). The conditioned plasma was then transfused to naïve mouse.

As shown on FIG. 2A, the <10 kDa fraction of the conditioned plasma frommouse having received mPEG allogeneic splenotytes retained the abilityto increase Treg levels in vivo. As shown on FIG. 2B, the <10 kDafraction of the conditioned plasma from mouse having received mPEGallogeneic splenotytes retained the ability to decrease Th17 levels invivo. The immunodulatory effect of conditioned murine plasma seems tomostly reside in the lower molecular weight fraction (<10 kDa). This lowmolecular weight fraction does not include the majority of cytokines(usually encompasses in the 100-30 and the 30-10 kDa fractions)typically thought to mediate immunodulation of Tregs andpro-inflammatory leukocytes. However, the <10 kDa fraction is suspectedto contain, among its components, microRNAs (miRNAs).

To determine if the miRNAs in the conditioned plasma mediated theimmunomodulatory effects observed with the conditioned plasma, mice wereinjected with control conditioned plasma or the same plasma that hadbeen pre-treated with RNase A, an enzyme that degrades/destroysribonucleic acids such as miRNAs. As noted in FIG. 2C, treatment withRNase A abolishes virtually all immunomodulatory activity of theconditioned medium, thereby confirming the ribonucleic acid nature ofthe size-fractionated conditioned plasma.

The size-fractionation conditioned plasma was administered to mice andits effects on the intracellular cytokine expression of CD4⁺ cells wasexamined. As shown on FIG. 3, the <10 kDa fraction and some of the <3kDa fraction of the conditioned plasma from mouse having received mPEGallogeneic splenotytes increase IL-10 intracellular expression in CD4⁺cells in vivo (FIG. 3A). However, the <10 kDa fraction and the <3 kDafraction of the conditioned plasma from mouse having received mPEGallogeneic splenotytes did not exhibit any increase in IL-2, TNF-α,IFN-γ or IL-4 intracellular expression in CD4⁺ cells in vivo (FIGS. 3Bto 3E). The <10 kDa (and some of the >3 kDa) fraction of the conditionedof the mPEG-allogeneic plasma, when compared to the correspondingfractions of the conditioned allogeneic plasma, increased the expressionof pro-tolerogenic cytokines, such as IL-10, while actively preventingthe expression of pro-inflammatory cytokines, such as IL-2, TNF-α, IFN-γor IL-4. Indeed, pro-inflammatory cytokines in the mPEG allogeneicplasma recipients remained at levels seen in naïve animals.

Example III—In Vitro and In Vivo Immune Modulation by miRNA-EnrichedAcellular Preparations

The conditioned plasma or the miRNA preparation (100 μL) obtained fromthe conditioned plasma (of mice having received saline, unmodifiedallogeneic splenocytes or polymer-modified allogeneic splenocytes) wereadministered intravenously to 7-8 week-old mice thrice (at days 0, 2 and4). Cohorts (n=4) of mice were sacrificed at days 30, 60, 120, 180 and270. Spleens were removed and CD4⁺ cells were stained for intracellularexpression of IL-2, IL-4, IL-10, INF-γ and TNF-α. Splenic Treg and Th17populations were also measured. As shown on FIGS. 4A-C, theadministration of the conditioned plasma or the derived miRNApreparation from mouse having received unmodified allogeneic splenocytescaused an increase in the expression of intracellular IL-2 and INF-γ inCD4⁺ cells. On the other hand, the administration of the conditionedplasma or the derived miRNA preparation from mouse having receivedmPEG-modified allogeneic splenocytes (i.e., TA1 preparation) caused anincrease in the expression of intracellular IL-10 in CD4⁺ cells. Thesemodulations in expression were observed until at least 270 days afterthe administration of the conditioned medium or the miRNA preparation.This data suggests that miRNA was an active component mediating theimmunological changes, RNase treatment of the conditioned plasma or ofthe miRNA preparation prior to administration to animals eitherdiminished (plasma) or abolished (miRNA) the immunomodulatory effects.While conditioned plasma retained some immunomodulatory effect, it isbelieved that it was due to residual cytokines and/or plasma-mediatedinactivation of the RNAase A enzyme.

As also shown on FIG. 4D, the administration of the conditioned plasmaor the derived miRNA preparation from mouse having receivedmPEG-modified allogeneic splenocytes (i.e., TA1 preparation) caused anincrease in the percentage of Treg (Foxp3⁺) cells in function of thetotal CD4⁺ cells. On the other hand, the administration of theconditioned plasma or the derived miRNA preparation from mouse havingreceived unmodified allogeneic splenocytes caused an increase in thepercentage of Th17 (IL-17⁺) cells in function of the total CD4⁺ cells(FIG. 4E). These modulations in CD4⁺ cells types were observed at least270 days after the administration of the conditioned medium or the miRNApreparations and were diminished (plasma) or abolished (miRNA) with apreliminary RNase treatment. Acellular preparations prepared from miceinjected with either allogeneic or mPEG-allogeneic leukocytes exertedpotent and long-lasting effects in naive recipient mice. In aggregate,allogeneic-derived preparations (plasma or miRNA) yielded apro-inflammatory state while mPEG-allogeneic-derived preparations(plasma or miRNA) yielded a immunoquiescent state.

Murine and human-derived miRNA preparations exert a direct effect oncell signaling. Murine TA1 preparations have been incubated with Jurkatcells (1×10⁶ cells/ml treated with 50 μl of TA1/ml) and the level ofphosphorylation of some of the phosphokinase has been measured after 30minutes of incubation. As shown on FIG. 5, TA1 preparations favored thephosphorylation of Akt and PRAS40 kinases while decreasing thephosphorylation of the HSP60 kinase.

Murine TA1 preparations were also introduced (at time 0) into a humanPBMC MLR assay in order to determine their effect on humanallo-recognition. As indicated on FIG. 6, the presence of the murine TA1preparations resulted in a dose-dependent decrease in the percentage inleukocyte proliferation (at both 10 and 14 days) which is indicative oftheir pro-tolerogenic effects. This data also indicates that the TA1preparations show significant evolutionary conservations (both sequencespecific and similarity) since the murine TA1 are highly effective in axenogeneic system (e.g. human MLR).

To compare the therapeutic efficacy of the manufactured miRNApreparations, the murine TA1 preparation was directly compared to aknown, clinically used, pro-tolerogenic therapeutic product (etanercept;trade name ENBREL®). TA1 and etanercept were introduced in a mouse MLR(using Blab/c and C57Bl/6 splenocytes) and the proliferation of thesplenocytes were measured. As shown in FIG. 7, TA1 more efficientlyrepressed CD4+ splenocyte proliferation (FIG. 7A) and CD8+ splenocyteproliferation (FIG. 7B) than did etanercept. This data demonstrates thatthe TA1 preparation induced a much more potent immunosuppressive effectthan the medicinal ingredient of the drug ENBREL®. While dosing ofCBS-TA1 is expressed in μl/ml, the active component (i.e., miRNA) withinthe TA1 preparation is in the pg-ng range.

While the murine TA1 preparation proved effective both in vitro and invivo in experimental models involving immunologically normal cells andanimals, to test the effectiveness of the TA1 preparation, a model ofautoimmune disease, NOD mice were used. In the NOD mice, autoimmunedestruction of the pancreatic islets begins within approximately 10-15weeks of birth and is confirmed by elevated blood glucose measures. Thelymphocytes from pre-diabetic and diabetic animals has been obtainedfrom the spleen, the brachial lymph node and the pancreatic lymph node.These lymphocytes have been submitted to flow cytometry usinganti-IL-17A (PE) and anti-FoxP3 (Alexa 697) antibodies. As shown in FIG.8, significant changes in the levels of Th17 and Treg lymphocytes arenoted in the spleen, brachial lymph node and pancreatic lymph nodes uponconversion of NOD mice from non-diabetic to diabetic state. Thesechanges are characterized by dramatically increased Th17 (top numbers ineach panels) and significantly decreased Treg (lower numbers in eachpanels) lymphocytes.

Murine TA1 preparations were obtained from normal Balb/c or C57Bl/6 miceand 100 μL was administered intravenously once to 10 week-old NOD mice(n=5). Naïve NOD mice were used for comparison. The administration ofTA1 caused a shift in immune modulation at day 5 post treatment towardsimmune tolerance by decreasing the circulating blood levels ofpro-inflammatory Th17 cells by over 50% (0.17% versus 0.38% foruntreated NOD mice).

As shown in FIG. 9A, the administration of the murine TA1 preparations(3 times 100 μl i.v. injections each 2 days apart) to 7 week-old NODmice NOD mice yielded significant protection against progression todiabetes. Results are shown as the percentage of normoglycemic animalsin function of age (in weeks) and treatment (dashed line=TA1, solidline=naïve NOD mice). In this model, diabetes begins to occur atapproximately 15 weeks. Between weeks 15 and 20, 75% of untreated micedeveloped hyperglycemia (i.e. diabetes) compared to 13% of TA1-treatedmice. After 30 weeks, 9 out of the 15 animals treated with TA1 remainednormoglycemic compared to only 4 out of the 16 for untreated animals.Even in the TA1-treated mice that developed diabetes (6 out of 15), TA1treatment significantly delayed the onset of diabetes with 67% of thediabetic animals occurring at greater than 20 weeks of age. In contrast,100% of the diabetic control NOD mice arose before 20 weeks of age.Moreover, the onset of diabetes correlated with the Treg:Th17 ratio asshown in FIG. 9B. A high Treg:Th17 ratio protected against, or delayed,the age of onset for overt diabetes. As shown, untreated diabetic NODmice demonstrated lower Treg/Th17 ratios compared to diabeticTA1-treated NOD mice. The higher Treg/Th17 ratio of the TA1-treated micesimilarly correlated with a delayed onset of diabetes in the mice thatdeveloped overt disease. At 30 weeks of age, all survivor (i.e.normoglycemic) mice were sacrificed and their Treg/Th17 ratiosdetermined As shown in FIG. 9B, very high Treg/Th17 ratio werecharacteristic of normoglycemic animals in both the untreated andTA1-treated groups. The importance of the Treg/Th17 ratio is furthershown in FIG. 9C in which the ratio is described in normal mouse strains(Balb/c and C57/Bl6) pre-diabetic NOD mice (7 weeks of age), diabeticcontrol and TA1-treated NOD mice as well as normoglycemic control andTA1-treated NOD mice. As shown by the normoglycemic animals, asignificantly higher (p<0.0001) Treg/Th17 ratio was observed relative todiabetic mice.

The administration of the murine TA1 preparations to NOD mice caused asystemic and/or local increase in pro-tolerogenic leukocytes. Leukocytepopulations were quantitated at time of sacrifice of the mice (weeks15-30). Treatment at 7 weeks of age with TA1 yielded a persistent andsignificant increase in Treg cells in all tissues of the NOD mousemeasured with the exception of the thymus (FIG. 10A). This data suggestthat TA1 exerts a potent immunomodulatory effect on lymphatic organs.Tregs counter-balance/attenuate proinflammatory lymphocytes such as Th17and Th1 cells. The administration of murine TA1 similarly caused anincrease in the expression of TGF-8. As shown in FIG. 10B, the TA1preparation increased TGF-β⁺ cells in the treated mice. TGF-βcounter-balance/attenuate proinflammatory lymphocytes such as Th17 andTh1 cells. Exogenous TGF-β has previously been shown to preventautoimmune diabetes in NOD mice. The administration of TA1s increasedthe level of expression of IL-4⁺, a marker of Th2 cells (FIG. 10C),further confirming its immunomodulatory effect on lymphatic organs. Theadministration of TA1s increased the level of expression of IL-10⁺,another marker of Th2 cells (FIG. 10D) as well as the percentage ofCD62L⁺ cells (FIG. 10E), CD152+ cells (FIG. 10F) and CD11c⁺ cells (FIG.10G). This modulation was however more pronounced in the pancreas of thetreated animals. Moreover, histopathological analysis demonstrated thatTA1 treatment prevented/diminished leukocyte infiltration anddestruction of the pancreatic islets (data not shown). While >95% ofislets examined from untreated NOD mice (both diabetic and 30 week oldnon-diabetic) exhibited overt insulitis (60%) or perinsulitis (40%),less than 10% of islets from the non-diabetic 30 week old TA1 miceexhibited overt insulitis while ˜60 percent of islets were completelynormal. In TA1-treated mice that became diabetic, approximately 35% ofislets were normal in appearance while 25% demonstrated overt insulitis,with the remained of mice exhibiting varying degrees of peri-insultis.

The administration of the murine TA1 preparations to NOD mice alsocaused a decrease in pro-inflammatory Th17 cells and Th1 cells, as shownby the decrease in the percentage of IL-17A⁺ cells (FIG. 11A) as well asthe decrease in the percentage of INF-γ⁺ cells (FIG. 11B), IL-2⁺ cells(FIG. 11C), TNF-α⁺ cells (FIG. 11D) and IL-12⁺ cells (FIG. 11E). Thisdata suggest that the TA1 preparations prevented Th17/Th1 upregulationin the treated mice. As it is know in the art, Th1 and Th17 lymphocytesmediate islet cell destruction. Interestingly, the administration ofTA1s caused a significant increase in the level of NK cells (as measuredby the expression of NK1.1+ cells on FIG. 11F) in the pancreas, but notin other tissues. It is believed that the differentially induced NKcells in the pancreas destroys autoreactive (i.e. inflammatory) cellsproviding an additional immunomodulatory mechanism resulting indecreased diabetes.

Further, it has been shown are that the administration of TA1s increasesB10⁺ (B regulatory) cells and tolerogeneic DC cell levels whiledecreasing APC associated with inflammation (data not shown) furtherconfirming the pro-tolerogenic effects of TA1 s.

Example IV—miRNA Characterization of Acellular ProtolerogenicPreparations

In order to characterize the constituents of the miRNA preparations, themiRNA of conditioned medium collected at 72 hours from resting humanPBMC, a human control MLR (using two HLA disparate PBMC populations),and a mPEG MLR (using the same two allogeneic PBMC populations whereinone population is modified with a polymer, e.g. mPEG) (this human miRNApreparation is herewith referred to as TA2) and compared via qPCRanalysis. The combined average of the resting Donor A and resting DonorB (i.e., resting AB) were used, unless otherwise noted, for baseline inall analyses.

As shown in FIG. 12A, when the miRNA population from the conditionedmedium from a control MLR is compared to the miRNA population of thesupernatant of resting cells, using a volcano plot analysis, at leastfive different miRNAs are differentially expressed (e.g. increased) bystatistical significance (p<0.01 for miR-9-5p, miR-155-5p, miR-206,miR-147a and p<0.05 for miR-214-3p) and at least one miRNA is modulatedby at least a log₂ (e.g. miR-302a-3p). In contrast, as shown in FIG.12B, when the miRNA population from the conditioned medium from a mPEGMLR (e.g. TA2) is compared, using a volcano plot analysis, with themiRNA population of the supernatant of resting cells, at least one miRNAis differentially expressed (e.g. increased) by statistical significance(p<0.05 for miR-214-3p) and at least one miRNA is modulated by at leasta log₂ fold (e.g. miR-149-5p). A direct comparison of mPEG-MLR (e.g.TA2) to the control MLR as shown in FIG. 12C, demonstrates that at leasttwo miRNAs are differentially expressed by volcano statisticalsignificance (p<0.01 for miR-155-5p and p<0.05 for miR-9-5p) and atleast two miRNAs are modulated by at least a log₂ (e.g. miR-183-5p andmir-147a).

On FIG. 12, nine miRNA species were identified. These miRNA species wereselected because they were considered to be differentially expressed asdetermined by clustergram analysis between the control MLR and mPEG-MLR.The miRNA species identified with 1, 2, 3, 5, 6, 8 and 9 showedincreased abundance in the mPEG MLR relative to the control MLR. ThemiRNA species identified with 4 has a relative abundance similar in boththe control MLR and mPEG-MLR and elevated relative to resting cells.

Further characterization of the miRNA population of the conditionedmedium of the control MLR and mPEG MLR is provided in fold changeanalysis. FIG. 13 provides a summary of the fold regulation of thepurified miRNA preparations differentially expressed in the conditionedmedium of a control MLR and a mPEG MLR (TA2) when compared to theconditioned medium of resting cells. FIG. 14 provides a subset of themiRNAs presented in FIG. 13 and exhibiting at least a log₂ foldmodulation when compared to resting cells. As indicated in FIG. 14, asubpopulation of miRNAs are decreased in the conditioned medium from themPEG MLR and increased in the conditioned medium from the control MLR(miR-183-5p, miR-203a, miR363-3p). As also indicated in FIG. 14, anothersubpopulation of miRNAs are increased in the conditioned medium from themPEG MLR and decreased in the conditioned medium from the control MLR(miR-21-5p, miR-27a-3p, miR27b-3p, miR-298, miR-34a-5p, let-7a-5p,let-7e-5p, miR-132-3p).

REFERENCES

-   Anderson M S, Bluestone J A. The NOD mouse: a model of immune    dysregulation. Annu Rev Immunol. 2005; 23:447-85.-   Bradley A J, Test S T, Murad K L, Mitsuyoshi J, Scott M D.    Interactions of IgM ABO antibodies and complement with    methoxy-PEG-modified human RBCs. Transfusion 2001; 41:1225-33.-   Bradley A J, Scott M D. Immune complex binding by immunocamouflaged    [poly(ethylene glycol)-grafted] erythrocytes. Am J Hematol 2007;    82:970-5.-   Chen A M, Scott M D. Current and future applications of    immunological attenuation via pegylation of cells and tissue.    BioDrugs 2001; 15:833-47.-   Chen A M, Scott M D. Immunocamouflage: prevention of    transfusion-induced graft-versus-host disease via polymer grafting    of donor cells. J Biomed Mater Res A 2003; 67:626-36.-   Chen A M, Scott M D. Comparative analysis of polymer and linker    chemistries on the efficacy of immunocamouflage of murine    leukocytes. Artif Cells Blood Substit Immobil Biotechnol 2006;    34:305-22.-   Le Y, Scott M D. Immunocamouflage: the biophysical basis of    immunoprotection by grafted methoxypoly(ethylene glycol) [mpeg].    Acta Biomater 2010; 6:2631-41.-   McCoy L L, Scott M D. Broad spectrum antiviral prophylaxis:    inhibition of viral infection by polymer grafting with    methoxypoly(ethylene glycol). In: PF T, editor. Antiviral drug    discovery for emerging diseases and bioterrorism threats. Hoboken, N    J: Wiley & Sons; 2005. p. 379-95.-   Murad K L, Gosselin E J, Eaton J W, Scott M D. Stealth cells:    prevention of major histocompatibility complex class II-mediated    T-cell activation by cell surface modification. Blood 1999A;    94:2135-41.-   Murad K L, Mahany K L, Brugnara C, Kuypers F A, Eaton J W, Scott    M D. Structural and functional consequences of antigenic modulation    of red blood cells with methoxypoly(ethylene glycol). Blood 1999B;    93:2121-7.-   O'Neill D W, Bhardwaj N. Differentiation of peripheral blood    monocytes into dendritic cells. Curr Protoc Immunol; 2005. Chapter    22: Unit 22F.4.-   Scott M D, Murad K L, Koumpouras F, Talbot M, Eaton J W. Chemical    camouflage of antigenic determinants: stealth erythrocytes. Proc    Natl Acad Sci USA 1997; 94:7566-71.-   Sutton T C, Scott M D. The effect of grafted methoxypoly(ethylene    glycol) chain length on the inhibition of respiratory syncytial    virus (RSV) infection and proliferation. Biomaterials 2010;    31:4223-30.-   Wang D, Toyofuku W M, Chen A M, Scott M D. Induction of    immunotolerance via mPEG grafting to allogeneic leukocytes.    Biomaterials. 2011 December; 32(35):9494-503.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A method of increasing a ratio of the level ofregulatory T (Treg) cells to the level of pro-inflammatory T cells in asubject in need thereof, said method comprising: (i) associating alow-immunogenic biocompatible polymer to a cytoplasmic membrane of afirst leukocyte to obtain a first modified leukocyte, wherein thelow-immunogenic biocompatible polymer is polyethylene glycol (PEG),2-alkyloxazoilne (POZ), or hyperbranched polyglycerol (HPG); (ii)contacting the first modified leukocyte with a second leukocyte underconditions to allow a pro-tolerogenic allo-recognition to provide aconditioned preparation, wherein the first leukocyte is allogeneic tothe second leukocyte; (iii) selecting miRNA components having anindividual average molecular weight of less than about 10 kDa from theconditioned preparation under conditions to inhibit RNA degradation by aRNAse and to maintain a relative abundance of each of the miRNAcomponents so as to obtain a composition enriched in acellularpro-tolerogenic components; (iv) formulating the composition of step(iii), under conditions to inhibit RNA degradation, in thepro-tolerogenic preparation for administration to the subject; and (v)administering to the subject a therapeutic amount of the pro-tolerogenicpreparation of step (iv); wherein the administration of thepro-tolerogenic preparation increases the ratio in the subject andwherein the increased ratio between the level of Treg cells and thelevel of pro-inflammatory T cells is for treating or alleviating thesymptoms associated to an auto-immune disease afflicting the subject. 2.The method of claim 1, where the process further comprises covalentlybinding the low-immunogenic biocompatible polymer to amembrane-associated protein of the cytoplasmic membrane of the firstleukocyte.
 3. The method of claim 1, wherein the low-immunogenicbiocompatible polymer is a polyethylene glycol (PEG).
 4. The method ofclaim 3, wherein the polyethylene glycol is a methoxy polyethyleneglycol (mPEG).
 5. The method of claim 4, wherein the process furthercomprises covalently binding the mPEG by contacting the first leukocytewith methoxypoly(-ethylene glycol) succinimidyl valerate.
 6. The methodof claim 1, wherein step (ii) of the process occurs in vitro.
 7. Themethod of claim 6, wherein the conditioned preparation is a supernatantof a cell culture of the first leukocyte and the second leukocyte. 8.The method of claim 6, wherein the process further comprises preventingone of the first leukocyte or the second leukocyte from proliferatingprior to step (ii).
 9. The method of claim 1, wherein step (ii) of theprocess occurs in vivo and comprises administering the first modifiedleukocyte to a mammal having the second leukocyte.
 10. The method ofclaim 9, wherein the conditioned preparation is plasma.
 11. The methodof claim 9, where the process further comprises preventing the firstleukocyte from proliferating prior to administration to the mammal. 12.The method of claim 1, wherein step (iii) of the process comprisesfiltering out components having the individual average molecular weightof more than about 10 kDa from the conditioned preparation.
 13. Themethod of claim 1, wherein step (iv) of the process comprisesformulating the composition for intravenous administration to thesubject.
 14. The method of claim 13, wherein step (v) of the processcomprises administering the composition intravenously to the subject.15. The method of claim 1, wherein the first leukocyte and/or the secondleukocyte is a T cell.
 16. The method of claim 15, wherein the T cell isa CD4-positive T cell.
 17. The method of claim 15, wherein the T cell isa CD8-positive T cell.
 18. The method of claim 1, wherein theauto-immune disease is at least one of type I diabetes, rheumatoidarthritis, multiple sclerosis, psoriasis, lupus, immunethrombocytopenia, experimental autoimmune encephalomyelitis, autoimmuneuveitis, inflammatory bowel disease, scleroderma and Crohn's disease.